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1885
Propranolol and Lidocaine Inhibit Neural
Norepinephrine Release in Hearts With
Increased Extracellular Potassium and Ischemia
Xiao-Jun Du, PhD; Rudolph A. Riemersma, PhD;
Keith A.A. Fox, MD; Anthony M. Dart, MRCP, PhD
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Background. Propranolol and lidocaine are effective antiarrhythmic drugs in myocardial ischemia and
infarction. As sympathetic nerve activation and norepinephrine release in ischemic hearts are arrhythmogenic, we tested the possibility that both agents inhibit neural norepinephrine release following
sympathetic activation in the ischemic environment.
Methods and Results. The model used was an in situ perfused innervated rat heart. Norepinephrine
release was induced by electrical stimulation of the left cervicothoracic stellate ganglion and analyzed
using radioenzymatic assay or high-performance liquid chromatography. In normoxically perfused
hearts, evoked norepinephrine release was not affected by either of the two agents at doses of 1 to 10
,gmol/L when extracellular K' concentration was 4 mmol/L but dose-dependently reduced at 10 mmol/L
K+ (D,L-propranolol: -53±4% at 1 ,umol/L and -64±6% at 10 gmol/L; lidocaine: -37±11% at 0.1
jzmol/L -67±5% at 1 ,umol/L, and -75±6% at 10 ,umol/L). At 10 mmol/L K', norepinephrine release
was not affected by timolol or atenolol (both 10 ,umol/L but was equally inhibited by D- or L-propranolol
at 10 ,umol/L (-56±5% and -53±9%o, respectively), indicating a P-blocking-independent mechanism. In
hearts with metabolic acidosis (pH 6.85) at K' of 4 mmol/L, neural norepinephrine release was also
reduced by propranolol at 10 pimol/L (-37%). Finally, in hearts perfused with 4 mmol/L K' and subjected
to 6-minute periods of ischemia, neural norepinephrine release was similarly suppressed by D,Lpropranolol (-38±6% at 0.1 ,umol/L, -445% at 1 ,zmol/L, and -78±3% at 10 ,umol/L) or lidocaine
(-39+7% at 0.1 umol/L, -58±9%o at 1 ,umol/L, and -91±3% at 10 ,umol/L).
Conclusions. These data indicate that propranolol and lidocaine inhibit neural norepinephrine release
via a Na+ channel-blocking mechanism that is synergistic with changes induced by ischemia, primarily
raised extracellular K'. This mechanism may contribute to the anti-ischemic and antiarrhythmic
properties of both agents in acute myocardial ischemia, which induces increased extracellular K' and
sympathetic activation. (Circulation. 1993;88[part 11:1885-1892.)
KEY WORDs * potassium * ischemia * nervous system * sodium * 3-adrenergic receptors
membranes
T he antiarrhythmic and anti-ischemic effects of propranolol in acute myocardial ischemia and infarction have been well established.1-4 In the setting
of ischemia, sympathetic activation with norepinephrine
(NE) release in the ischemic myocardium is considered of
importance in mediating ventricular arrhythmias.5 The
protective effects of 3-adrenergic antagonists are generally
attributed to blockade of 8-adrenoceptors on myocytes.
Whether the antiarrhythmic effect may be partly due to
inhibition of neural norepinephrine release in the ischemic myocardium is unknown. Although it is well known
that activation of presynaptic f3-adrenoceptors facilitates
norepinephrine release, studies examining the effect of
P-antagonists on norepinephrine release yield conflicting
results.6 In addition, some P-antagonists, like propranolol,
Received March 11, 1993; revision accepted June 22, 1993.
From the Cardiovascular Research Unit (R.A.R., K.A.A.F.),
Department of Medicine (RIE), University of Edinburgh, Edinburgh, UK; and Baker Medical Research Institute (X.-J.D.,
A.M.D.), Melbourne, Australia.
Correspondence to Dr X.-J. Du, Baker Medical Research
Institute, Prahran, Victoria 3181, Australia.
possess a nonspecific membrane stabilizing activity, in-
cluding the blockade of voltage-gated Na' channels.7 This
activity, however, is generally considered of minor importance, as it requires concentrations far above those
achieved clinically.
Acute myocardial ischemia induces regional hyperkalemia and acidosis within minutes of interruption of
coronary flow.8'9 Elevated extracellular K' concentration ([K+]o) is known to interfere with the conduction of
the action potential in Purkinje fibers or myocytes9-13
and perhaps also in adrenergic nerves.14 Interestingly,
studies in vitro have revealed a potentiation, by a raised
[K']. or by acidosis, of inhibitory effects of lidocaine on
Na' channels of myocardium.10-12,15,16 As depolarization
of the neural plasmalemma by Na' influx via voltagegated Na' channels is necessary for action potential
propagation and subsequent norepinephrine release,17
an inhibition of Na' channels of the neural membrane
in ischemia may suppress norepinephrine release and
hence the intensity of the subsequent adrenergic stimulation to the ischemic myocardium. This possibility,
however, has never been tested.
1886
Circulation Vol 88, No 4, Part 1 October 1993
Using a perfused, innervated rat heart model,1819 we
have therefore studied the effects of propranolol and
lidocaine on neural norepinephrine release during simulated ischemia (ie, increased [K']. and acidosis) and
stop-flow ischemia. We chose lidocaine as a reference
agent for propranolol because it is a well-defined Na'
channel blocker with effects known to be enhanced by
increased [K']. or by acidosis'0-1215,16 and also because
it is an effective agent for the acute treatment of
ischemic arrhythmias.20
Methods
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Preparation
Male Sprague-Dawley rats (280 to 350 g) were used
for this study. Experiments were carried out using a
perfused, innervated rat heart model previously described in detail.18"19 Rats were anesthetized with pentobarbital (60 mg/kg IP) and heparinized (200 U per rat
IV). The chest was opened and a metal cannula inserted
into the ascending aorta to start coronary perfusion in
situ. The perfusate was a modified Krebs-Henseleit
solution containing (in mmol/L) Na' 145, K' 4.0, Ca2
1.85, Mg2- 1.05, HCO3- 25, P043- 0.5, glucose 11, and
EDTA 0.027. The buffer was continuously gassed with
95% 02-5% CO2 (pH 7.4) and warmed to 37°C. Perfusion flow rates were controlled by a peristaltic pump and
set at 5 mL* g` * min' by the estimated heart weight.
The left cervicothoracic stellate ganglion, with the
cardiac nerves attached, was separated and mounted on
a pair of bipolar electrodes for subsequent electrical
stimulation using a model S88 or a model SD9 stimulator (Grass Instrument Co, Quincy, Mass).'9 The nerves
were continuously superfused with warm and oxygenated buffer except when stimulated. Stimuli had a pulse
width of 2 milliseconds, a current of 0.8 mA, and a
frequency of 5 Hz.
After ligation of bilateral pulmonary vessels and the
superior vena cava, a catheter was inserted via the
inferior vena cava into the right atrium for the collection
of coronary venous effluent. The recovery of coronary
effluent was between 85% and 100%.
The left ventricle was cannulated via the apex, and
the ventricular pressure was derived from a pressure
transducer (Elcomatic, Glasgow, UK) or a microtip
catheter pressure transducer (model SPR-249, Millar
Instruments Inc, Houston, Tex) and was recorded on a
TA2000 recorder (Gould Inc, Cleveland, Ohio) or a
model 7 polygraph (Grass Instrument). Heart rate was
measured from ventricular pressure traces.
Alteration of K' levels in the perfusate was achieved
by infusion, via a model 22 or a model 901A pump
(Harvard Apparatus, South Natick, Mass), of KCl solution, and final K' concentrations achieved were ascertained by measuring K' levels in the perfusate and in
the venous effluent collected in the absence of nerve
stimulation using a model 501 Na/K analyzer (Instrumentation Laboratory, Milan, Italy). Global heart ischemia was induced by stopping of perfusion, and the
myocardial temperature was kept at 37°C by covering
hearts with a thermostatic chamber. Coronary effluent
was collected during the first 2 minutes of the restoration of coronary flow to the preischemic level.
Norepinephrine Assay
Two norepinephrine analysis methods-radioenzymatic assay and high-performance liquid chromatography (HPLC) with electrochemical detection -were used
in this study. Samples collected from one experiment
were always assayed with the same method and, whenever possible, in a single assay run. In experiments
carried out in Edinburgh, effluent samples were immediately cooled on ice and mixed with an equal volume of
perchloric acid (final concentration, 0.3N) and stored at
-40°C until assayed. Concentrations of norepinephrine
were analyzed radioenzymatically in duplicate, and the
average of the two measurements was used.2' The
intra-assay coefficient of variation at 2 pmol/mL was
7%. For those experiments carried out in Melbourne,
effluent samples were immediately frozen on dry ice and
stored at -70°C until analyzed using an HPLC method.
Norepinephrine was extracted using alumina adsorption, separated by HPLC, and quantified by electrochemical detection.22 The intra-assay coefficient of variation was 3%.
Drugs Used
Desipramine, atenolol, timolol, D,L-propranolol,
D-propranolol, L-propranolol (all from Sigma Chemical
Co, St Louis, Mo), butoxamine (provided by Dr A.
Ungar, Department of Pharmacology, University of
Edinburgh), and lidocaine chloride (Delta West Ltd,
Bentley, Australia) were used.
Protocols
A 20-minute period of perfusion with normal perfusate was allowed to stabilize preparations before the
experiment. All experiments were carried out in the
presence of desipramine (final concentration of 0.1
,umol/L) to inhibit neural norepinephrine reuptake.
Sympathetic ganglion stimulation was performed at 5
Hz for periods of 30 or 60 seconds with 15-minute
intervals between stimuli. The first stimulus (S,) served
as a reference for each experiment. Drugs or KCl were
infused into the heart at least 10 minutes before the
subsequent stimulation. Coronary effluent was collected
for a period of 2 minutes immediately before or during
and after nerve stimulation. In two groups of hearts
(n=8 each) perfused with either 4 or 10 mmol/L K+, the
reproducibility of norepinephrine release in response to
five episodes of nerve stimuli (30 seconds) was
examined.
Propranolol and norepinephrine release. The effect of
propranolol and other P8-antagonists on norepinephrine
release was examined in seven separate groups of hearts
perfused with various perfusate K' concentrations (Table 1). Hearts in group 1 were initially perfused with 4
mmol/L K'. After a control nerve stimulus (S,, 30
seconds), D,L-propranolol was infused into the heart at
1 ,umol/L throughout the subsequent experiment, and
another four stimuli (30 seconds each) were performed
at K+ concentrations of 4 (S2), 7 (S3), 10 (S4), and 13
mmol/L (S5), respectively. In group 2, hearts were
perfused with 10 mmol/L K+, and four nerve stimuli (30
seconds each) were given in the absence (S,) and
presence of D,L-propranolol at 0.1 (S2), 1 (S3), and 10
,gmol/L (S4), respectively.
Du et al Propranolol, Lidocaine, and Norepinephrine Release
TABLE 1. Protocols for Experiments
on Neural Norepinephrine Release
1887
Examining the Effect of Propranolol and Other P-Antagonists
Sequence of Nerve Stimulation (5 Hz, 30 seconds)
Group
1 (n=7)
2
3
4
5
6
7
S,
4/10/10/10/10/10/-
S2
4/1 (D,L-propranolol)
S3
7/1 (D,L-propranolol)
10/1 (D,L-propranolol)
10/5+5 (L+D-propranolol)
10/5+5 (D+L-propranolol)
10/10 (timolol)
S4
10/1 (D,L-propranolol)
10/10 (D,L-propranolol)
S5
13/1 (D,L-propranolol)
10/0.1 (D,L-propranolol)
10/10 (L-propranolol)
...
10/10 (D-propranolol)
...
10/10 (atenolol)
...
10/10 (butoxamine)
10/10 (D,L-propranolol)
...
4/4/10 (D,L-propranolol)
...
...
Values in the table denote concentrations of perfusate K+ in mmol/L and agents tested in ,umol/L (mmol/Lpmol/L).
(n=8)
(n=8)
(n=8)
(n=7)
(n=8)
(n=7)
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The effect of D- and L-isomers of propranolol on
norepinephrine release was tested in groups 3 and 4
with perfusate K' of 10 mmol/L. The first control
stimulus (S,, 30 seconds) was followed by another two
stimuli (30 seconds each) in the presence of D- or
L-propranolol (10 ,umol/L, respectively, S2) or a combination of D- and L-propranolol (5 ,umol/L each, S3).
In groups S and 6, hearts were perfused with 10
mmol/L K', and three stimuli (30 seconds each) were
performed in the absence (S,) and presence (S2 and S3)
of 18-antagonists. The agents presented at S2 and S3 were
atenolol (10 ,umol/L, S2) and timolol (10 ,umol/L, S3) for
group S and butoxamine (10 ,umol/L, S2) and D,Lpropranolol (10 1tmol/L, S3) for group 6. In comparison,
hearts in group 7 were perfused with 4 mmol/L K', and
sympathetic ganglion was stimulated twice (30-second
duration each) without (S,) and with (S2) D,L-propranolol at 10 ,umol/L.
The protocols for this series of experiments are also
summarized in Table 1 for clarity.
Lidocaine and norepinephrine release. The effect of
concentrations of lidocaine on norepinephrine release
was examined in two groups of hearts (n=7 each)
perfused with 4 or 10 mmol/L K', respectively. Nerve
stimulation was performed five times (30 seconds each)
in the group perfused with 10 mmol/L K' or six times
(30 seconds each) in the group perfused with 4 mmol/L
K+. The first stimulation served as control (S,), and
concentrations of lidocaine tested were 0.1 (S2), 1 (S3),
10 (S4), 100 (S5), and 400 ,umol/L (S6), respectively.
Propranolol, lidocaine, and norepinephrine release with
low pH. Effects of D,L-propranolol and lidocaine on
norepinephrine release at a reduced pH were examined
in three groups of hearts (all n=7) perfused with 4
mmol/L K'. The perfusate pH was reduced by lowering
NaHCO3 from 25 mmol/L to 7 mmol/L, and NaCl
concentration was increased accordingly. The final pH
was adjusted to 6.8 to 6.9 (average, 6.85) by the addition
of concentrated HCl. In all three groups, the first nerve
stimulus (S,, 1 minute) was performed at normal pH of
7.4. Then the perfusate pH was reduced to 6.85, and the
second and third stimuli (1 minute each) were applied
after 10 (S2) or 25 minutes (S3) of perfusion with low pH
buffer. Group 1 served as the control, and D,L-propranolol (group 2) or lidocaine (group 3) was presented at S2
(1 ,umol/L) and S3 (10 ,umol/L), respectively.
...
...
...
...
...
...
Propranolol, lidocaine, and norepinephrine release in
ischemia. Effects of D,L-propranolol and lidocaine on
norepinephrine release in ischemic hearts were tested.
Five groups of hearts (n=7 to 10) were perfused with 4
mmol/L K' and subjected to three periods of 6-minute
total ischemia separated by 15-minute intervals of perfusion at 5 mL- g- min- . Nerve stimulation (5 Hz, 1
minute) were performed in the final minute of ischemic
periods. Group 1 served as control and received no drug
treatment. In the remaining four groups, propranolol
(two groups) or lidocaine (two groups) were infused 10
minutes before the second and third periods of ischemia
(0.01 and 0.1 pimol/L for one group and 1 and 10
,umol/L for one group, respectively). Previous studies
have shown that there is no ischemia-induced norepinephrine release by periods of total ischemia less than
10 minutes.23
Statistical Analysis
Results are expressed as mean±SEM. Whenever
possible, each animal served as its own control to
eliminate between-animal variation in quantitative norepinephrine release and to improve statistical power.
Therefore, norepinephrine data are presented both in
absolutes and in percentage of individual values measured before and after an intervention. Differences
were tested for statistical significance by one- or twoway ANOVA, followed by paired- or unpaired Student's
t test. Bonferroni's correction was performed for comparison of repetitive measurements between groups.
P<.05 was considered significant.
Results
Basal norepinephrine release was always low (0 to 1.3
pmol* g`. min') and not influenced by perfusion with
increased K' concentrations (4 mmol/L: 0.7+0.2
pmol* g.* min-1, 10 mmol/L: 1.0+0.1 pmol * g`-* min'.
P=NS). In control hearts, quantities of norepinephrine
release evoked by S1S5 were not significantly different
within or between groups (P=NS by ANOVA or paired t
test: 4 mmol/L K', 34.0±3.9, 36.2+4.1, 33.8+4.6,
34.4±5.8, and 33.5+±5.5 pmol/g, respectively; 10 mmol/L
K+, 37.1+5.0, 39.9±6.2, 34.6+5.8,33.1±4.8, and 31.9±4.3
pmol/g, respectively). The percentages of S2SS over S,
(individual control) were 108±4%, 99±7%, 97±10%, and
95 +8%, respectively, for the group with 4 mmol/L K' and
107+7%, 91+±6%, 89+5%, and 86+6%, respectively, for
Circulation Vol 88, No 4, Part 1 October 1993
1888
i
50
Propranolol (1 FM)-
60
40-
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30 -
,40
30
20.-
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4
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Potassium concentration (mM)
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nAv
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0
0.1
1
20.
0 Si
10
0
*5E 3
E*S2
C D D+L
C L L+D
C A T
C B P
C P
FIG 2. Bargraph of effects of -adrenoceptorantagonists (all
at 10 munolIL) and stereoisomers of propranolol (both at S
pimol/L) on norepinephrine (NE) release in response to nerve
stimulation in hearts perfused with 4 mmolIL or 10 mmolIL
K+. Sympathetic ganglion was stimulated at 5 Hz for a period
of 30 seconds. n = 7 or 8 per group. D indicates D-propranolol;
L, L-propranolol; C, control; A, atenolol; T, timolol; B, butoxamine; and P, D,L-propranolol. * P<.05 and **P<.I001 versus
control values in the same group by paired t test.
[K+J 10mM
z 40
he
4mM
1
9
.50
he
0.
[K'1
[K-] 10 mM
|
10
Propranolol (AM)
FIG 1. Top, Plots of influence ofperfusate K' concentration
on the inhibition of nerve stimulation (5 Hz for 30 seconds)
mediated norepinephrine (NE) release by D,L-propranolol at 1
jumol/L (n= 7). Bottom, Plot of inhibition by varying concentrations of DL-propranolol of norepinephrine release induced
by sympathetic ganglion stimulation (5 Hz for 30 seconds) in
hearts perfused with 10 mmol/L K' (n =8). *P<.01 versus the
values by control stimulation (S,, without propranolol) by
paired t test.
-
the group with 10 mmol/L K' (P=NS for within- and
between-group comparisons).
Propranolol and Norepinephrine
Release at Increased K'
DL-Propranolol at 1 ,umol/L failed to modify norepinephrine release induced by nerve stimulation during
perfusion with 4 mmol/L K' but showed a progressively
enhanced inhibition of such release with increasing
perfusate K' from 7 to 13 mmol/L (F=6.91, P<.01 by
ANOVA, 7 mmol/L: -44±11%, 10 mmol/L: -50±7%,
and 13 mmol/L: -59±9%, all P<.01 by paired t test
versus Si, Fig 1). At 10 mmol/L perfusate K', inhibition
of norepinephrine release by propranolol was dose
dependent (F=25.8, P<.001; 0.1 1£mol/L: -7.7±10.3%,
P=NS; 1 Mimol/L: -53.5±4.0%, P<.01; and 10 ,umol/L:
-63.7±6.0%, P<.001, Fig 1).
At 4 mmol/L K', D,L-propranolol of 10 limol/L was
ineffective in modifying norepinephrine release with an
S2/S1 ratio of 110±8%. With 10 mmol/L K', norepinephrine release was not influenced by atenolol or
timolol at 10 gmol/L but was inhibited moderately by 10
,umol/L butoxamine (-32±9%, P<.05) and markedly
by 10 gmol/L D,L-propranolol (-69±5%, P<.001, Fig
2). Administration of D- or L-propranolol alone significantly suppressed norepinephrine release to a similar
extent to that observed by the combination of the two
isomers (-55±5% versus -59±8% and -53±9% versus -59±8%, both P=NS, Fig 2). There was a good
correlation between the reduction of norepinephrine
release produced by either of the two isomers and that
by simultaneous infusion of both isomers (r=.80, P<.01,
n=16).
Lidocaine and Norepinephrine Release at 4 and 10
mmol/L K'
In hearts perfused with 4 or 10 mmol/L K', basal
norepinephrine overflow remained low and not affected
by lidocaine even at 100 1umol/L (0.68±0.22 versus
1.01+0.11 pmol*g 1 min', P=NS). There was no
significant difference in norepinephrine release by S, in
groups with 4 or 10 mmol/L K' (49.3+6.7 versus
41.2+9.5 pmol/g). With 4 mmol/L K', norepinephrine
release in response to nerve stimulation was not significantly affected by lidocaine until the concentration was
10 ,tmol/L or above (P<.02, Fig 3). In contrast, in hearts
with 10 mmol/L K', there was a dose-dependent suppression of norepinephrine release by lidocaine that was
statistically significant starting at 0.1 limol/L (F=8.54,
P<.001 by two-way ANOVA for overall difference at a
dose of 0.1 to 100 ,tmol/L, Fig 3). The average dose
required to suppress norepinephrine release by 50% of
control (IC50) was about 70 ,umol/L with 4 mmol/L K'
and 0.5 ,umol/L with 10 mmol/L K'.
Effects of Propranolol and Lidocaine on Basal Heart
Rate at 4 and 10 mmol/L K'
Basal heart rate remained stable in hearts perfused
with 4 or 10 mmol/L K' (244+6 and 232±9 beats per
minute, P=NS, combined data from 20 and 47 hearts,
respectively). Both D,L-propranolol and lidocaine reduced basal heart rate, in a concentration-dependent
manner, with 4 or 10 mmol/L K'. Interestingly, for both
agents, the dose-response curves of heart rate reduction
were similarly shifted leftward by an increased perfusate
K+ concentration (Fig 4). At 10 mmol/L K', D- and
L-isomers of propranolol at 10 gmol/L reduced basal
heart rate to a similar extent to that produced by 10
11mol/L D,L-propranolol (-162±+6 and -151±8 versus
-165±6 beats per minute, P=NS).
Propranolol, Lidocaine, and Norepinephrine Release:
Effect of Metabolic Acidosis
Norepinephrine release evoked by the first stimulus
(S,) at normal pH was similar in all three groups. In the
Du et al Propranolol, Lidocaine, and Norepinephrine Release
1889
100.
80-
05
0
U
z
El
60-
rw
z
40-
si
S2
a
~.
--0-
Contl
Pmpranlol
Lio
200
1
pH74a
pH 7.40
H68
pH 6.85
(10 mmn)
*
~j
X
S3
pH6.5
pH 6.8S
(25 min)
FIG 5. Plot of effects of DL-propranolol and lidocaine on
evoked norepinephrine (NE) release in perfused hearts with
metabolic acidosis (pH reduced from 7.40 to 6.85, K+ =4
mmol/L). The sympathetic ganglion was stimulated (5 Hz for
1 minute) once at a perfusatepHof7.4 (S,) and a further two
times after 10 and 25 minutes ofperfusion at a pHof 6.85 (S2
and S3, respectively). Propranolol or lidocaine were presented
10 minutes before and during 52 at 1 limol/L and 10 minutes
before and during 53 at 10 ,umol/L. n=7per group. *P<. 05
and 'P <. 0 versus individual values at pH 7.4 (S,) in the
same group by paired t test.
0
U
Wo
z
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
Lidocaine (-Log M)
FIG 3. Plots of effects of varying concentrations of DLpropranolol or lidocaine on norepinephrine (NE) release
induced by sympathetic ganglion stimulation (5 Hz for 30
seconds) in hearts perfused with 4 mmol/L or 10 mmol/L K+.
Results are presented as percentages of the individual values of
NE release without drugs (S,). *Denotes a significant difference between the 4 and 10 mmol/L K' groups by unpaired t
test with Bonferroni's correction. n = 7 or 8 in each group.
Propranolol, Lidocaine, and Norepinephrine Release
in Myocardial Ischemia
In hearts preperfused with 4 mmol/L K' and subsequently undergoing three periods of 6-minute total
ischemia, norepinephrine release evoked by nerve stimulation was reproducible in the control group (Table 2).
Treatment with DL-propranolol or lidocaine before
ischemia resulted in a significant and dose-dependent
inhibition of norepinephrine release starting at a concentration of 0.1 limol/L versus S, without drug (Table
2 and Fig 6). In the control group, each nerve stimulation (S1S3) during ischemia induced a significant increase in heart rate (+32±9, +32±9, and +36±10
beats per minute, respectively). This chronotropic response to nerve stimulation in the ischemic heart was
partly or totally inhibited by lidocaine and propranolol
(data not shown).
control group, norepinephrine release was not affected
by 10-minute perfusion (104±7% of S,, P=NS) but was
reduced by 25-minute perfusion with the acidic buffer
(79±t8% of S,, P<.05, Fig 5). In groups receiving drug
treatment, DL-propranolol or lidocaine at 1 ,umol/L did
not reduce norepinephrine release after 10-minute
acidic perfusion (101±6% or 93±5% of S,, respectively,
all P=NS). A 25-minute acidic perfusion together with
propranolol of 10 limol/L suppressed norepinephrine
release to 43±7% of S, (P<.01), a value significantly
lower than the control group without drug treatment
(P<.01). In the presence of lidocaine at 10 Mmol/L,
norepinephrine release by S3 tended to be lower than
the control group (56±10%, P=.09), but this was statistically insignificant. Acidic perfusion profoundly suppressed the systolic ventricular pressure during basal
and nerve stimulation (data not shown).
Discussion
The present study demonstrates a dose-dependent
inhibition of neural norepinephrine release by propranolol only at an increased [K']. and, to a lesser extent, at
a reduced extracellular pH. Lidocaine, a specific Na'
channel blocker, shows a very similar inhibitory effect
on norepinephrine release with a marked potentiation
240
a
200
.0
160
-0-
4mM K+
10mM K+
FIG 4. Plots of heart rate-lowering
activity of DL-propranolol and
idocaine and the potentiation by increasingK+ concentration from 4 to 10
mmoliL. Note the similar leftward
shift of the dose-response curves for
both agents by 10 mmol/L K' (arrows). HR indicates heart rate.
/
804
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40.
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0
0
0.1
1.0
10
Concentration of propranolol (piM)
0
0.1
1
10
100
Concentration of lidocaine (gM)
1000
Circulation Vol 88, No 4, Part 1 October 1993
1890
TABLE 2. Effects of D,L-Propranolol (0.01 to 10 jumol/L) and Lidocaine (0.01 to 10
gmol/L) on the Neural Norepinephrine Release (pmollg) in Hearts Perfused With 4
mmol/L K+ and Subjected to 6-Minute Periods of Total Ischemia
Group
1 (n=7)
2 (n=9)
Drug
None
Propranolol
3 (n=10)
Propranolol
4 (n=9)
Udocaine
5 (n=8)
Udocaine
Nerve stimuli (S1S3) were applied
treatment.
*P<.001 vs
S1
33.0+5.2
43.8+3.1
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r1-rT
1T
80
oo
a 0.01 gM
*
0.1 pM
10LM
*
10~±M
*
20
*
40.
1-
z
27.7+4.3
38.3±4.0
(0.01 ,umol/L)
S1 in the same group by paired t test.
Mechanism of the Inhibition of Norepinephrine
Release by Propranolol or Lidocaine at High [K'],
Several mechanisms could account for the inhibition
of neural norepinephrine release by propranolol. As
this inhibition is observed only at raised [K'], of 7 to 13
mmol/L, an inhibition by high K' of norepinephrine
release is possible. Although in some models hyperkalemia alone can inhibit evoked norepinephrine overflow
and postsynaptic responses,14,2425 previous studies and
the present study with this model show no effect on
norepinephrine release by increasing perfusate K' up to
13 mmol/L.19 Blockade of facilitatory presynaptic R2adrenoceptors could lead to a reduced norepinephrine
release.6 In this model with a normal K', however,
propranolol fails to suppress norepinephrine release
-
S3
30.2±4.5
27.3±3.6*
(0.1 ,umol/L)
31.0+4.3
12.7±2.9*
5.8±0.6*
(1 limol/L)
(10 gmol/L)
45.3±5.7
33.7+5.7
28.1+±5.2*
(0.1 ,umol/L)
(0.01 umol/L)
46.4+6.8
17.4±4.6*
3.1+±0.4*
(1 ,umol/L)
(10 ,umol/L)
in the last minute of ischemic periods. S1 served as a reference without drug
by raised [K'].. Finally, norepinephrine release in ischemic hearts is dose dependently suppressed by both
agents.
100-
S2
0
20
Propanolol
Ldocaine
FIG 6. Bar graph of inhibition of neural norepinephrine
(NE) release by concentrations of DL-propranolol and
lidocaine in the ischemic heart. Data have been presented as
percentages of the NE overflow by a control nerve stimulation
in the absence ofdrug treatment (S,=100%) calculated from
the absolutes presented in Table 2. In the control group
(n = 7), there was no significant difference in the amount of
NE released by S2 and 53 compared with that by S,
(92.8±7.0% and 102.3+13.7% of 5,, respectively). Note the
dose-dependent inhibition of NE release by both agents in the
ischemic conditions. *P<.001 versus individual control values
released by S, in the same group (paired t test).
according to our previous and the present studies.18
Further evidence against such a possibility comes from
the findings that the inhibitory effect of propranolol is
not shared by timolol, a potent nonselective (-blocker,
and that the two isomers of propranolol, with and
without 13-blocking activity, are equally effective in the
suppression of norepinephrine release at an increased
[K'].. Finally, the inhibition of norepinephrine release
by lidocaine is also potentiated by a raised [K']., and
this again does not indicate a presynaptic f3-adrenergic
mechanism. A mild inhibition of norepinephrine release
was observed by butoxamine, a relatively selective 82antagonist, at 10 mmol/L K'. This is probably due to
effects other than blockade of presynaptic P2-adrenoceptors, and the properties of butoxamine are still only
partly understood.
Propranolol also possesses membrane stabilizing activity, including the inhibition of Ca2' and Na+ channels.7 Theoretically, blockade of either channel (N-type
Ca21 channels for neuronal tissues) could lead to an
inhibited neurotransmission, as action potential propagation along the axons is mediated by Na+ influx and
norepinephrine exocytosis is the result of Ca21 influx at
the nerve varicosities. The dependency of the effect of
propranolol on [K']. suggests that Ca21 channels are
not involved as they function at near-zero membrane
potential and therefore should not be sensitive to
increased [K']. to the levels studied. In contrast, the
functional state of Na+ channels depends on the resting
membrane potential, which is in turn determined by the
K' gradient across the membrane.9 The similar effect of
lidocaine on both norepinephrine release and heart rate
at increased [K']. provides further support for a Na+
channel-dependent mechanism. In our study, propranolol is also synergistic with an increased [K']. in the
suppression of the basal heart rate. Sinus automaticity is
determined by the rate of spontaneous depolarization of
the resting membrane potential,26 and the main ionic
currents involved in this pacemaking are a background
inward Na+ current (IbNa) and a diminishing outward K'
current (hk).26 Thus, an inhibition of the Na+ inward
current may suppress the automaticity of the sinoatrial
node.
Du et al Propranolol, Lidocaine, and Norepinephrine Release
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
The mechanism for the inhibition of norepinephrine
release with raised [K']. may relate to changes in the
functional state of Na' channels with a resultant change
in the affinity of the channel for the two drugs. Na'
channels exist in three functional states: resting (R),
open (0), and inactivated (I).27-30 Blockade of Nal
channels by drugs is more likely when the channel is in
I form, perhaps due to an increased affinity and accessibility of agents to channel-binding sites.27-30 According
to the K' equilibration potential Ek, an increase in [K'].
from 4 to 10 mmol/L may lead to a 20-mV depolarization of the resting membrane potential,9 and this could
result in an inactivation of about 90% of Na' channels.
There is evidence that lidocaine binds mainly to Na'
channels in I form, thereby blocking channels and
inhibiting the transition of functional states from I to R.
In addition, after binding with lidocaine, channel reactivation requires a much more negative membrane
potential.27-30 This is largely prevented by a high [K+]0induced partial depolarization, leading to a progressively increased channel blockade. Studies have indeed
shown that an increased [K']. enhances the depressant
effects of lidocaine and other class I antiarrhythmic
agents on the electrophysiology of myocytes.10-12'28
Thus, we speculate that a synergistic inhibition of Na'
channels by propranolol or lidocaine and an increased
[K']. is the ionic mechanism for the inhibition of
norepinephrine release observed. Further studies are
required to confirm this mechanism in neuronal
preparations.
Contribution of Metabolic Acidosis
Reduction of external pH to 6.85 for 25 minutes had
only a minor effect on neural norepinephrine release, in
keeping with our previous observation.31 At the reduced
pH, propranolol at 10 1£mol/L reduced norepinephrine
release by 37%, although no such synergism was observed
at 1 ,umol/L. The same tendency was also found for
lidocaine. This could be due to both higher drug concentration and development of intracellular acidosis, via an
enhanced H+/K' and inhibited H+/Na+ exchange,9'32 following longer acidotic perfusion. Potentiation by acidosis
of the electrophysiological effects of lidocaine has been
previously demonstrated.15"1628 Reduction in pH reduces
Vma. and the resting membrane potential, which is due to
an enhanced K' efflux via H+/K' exchange and a suppressed Na+/K+ ,ATPase.9"11,1516,28 Following reduction in
the resting membrane potential, the fraction of inactivated
Na+ channels may increase and hence the blockade of the
Na+ channel by drugs."1128 In addition, acidosis lengthens
the action potential duration, which may increase the time
constant of recovery for the blocked and inactivated
channels.1128 The present study provides data showing
that the synergism of acidosis and Na+ channel blocker,
observed previously in Purkinje fibers or myocytes, also
pertains to the presynaptic adrenergic nerves.
Inhibition of Neural Norepinephrine Release in
Ischemic Heart
Acute ischemia rapidly leads to an increase in [K'].
and acidosis,8'9 and propranolol has no effect on the rise
in [K+]0.8,33 Our results indicate that propranolol and
lidocaine, while having little effect on norepinephrine
release under physiological conditions, show a potent
and dose-dependent inhibition of neural norepineph-
1891
rine release in the ischemic heart with a threshold
effective dose of 0.1 ,umol/L. The extent of the inhibition of norepinephrine release by either propranolol or
lidocaine at 10 ,mol/L is more pronounced in ischemia
(-88.5% and -90%, respectively) than during normoxic perfusion with 10 mmol/L K' (-59---69% for
propranolol and -75% for lidocaine), suggesting that
additional factors in ischemia, such as acidosis and
hypoxia,"1"15"16,28'34 may also contribute to the observed
drug effect. Our experiments with acidosis support this
view. However, a raised [K']. is by far the most important component.
Interestingly, our results are in keeping with the
findings from a study in in vivo rats with coronary artery
occlusion.35 In that study, the myocardial norepinephrine content was unchanged, but a marked reduction in
the density of fluorescing adrenergic fibers was found in
the ischemic area after 60 minutes of local ischemia,
indicating a local release and accumulation of norepinephrine in the ischemic zone. These changes could be
partly or completely prevented by pretreatment with
different doses of lidocaine.35 Our study provides direct
evidence for the mechanism of this inhibited norepinephrine release in vivo.
Clinical Implications
Propranolol and lidocaine are effective in the suppression of malignant arrhythmias and cellular damage in the
acute phase of myocardial infarction,'-5 20 and adrenergic
involvement in these processes has been supported by a
large number of studies.5,9,19 However, few of the previous
studies have linked these therapeutic effects with norepinephrine release in the ischemic myocardium. The inhibition of neural norepinephrine release demonstrated in
this study is likely to contribute to the therapeutic effects
of propranolol and lidocaine in myocardial ischemia. This
may also be the case for other class I and III antiarrhythmic agents. Our data may also help to explain the clinical
findings that the class I antiarrhythmic agents, although
particularly effective in suppressing ischemic arrhythmias,
are much less effective in the attenuation of ventricular
arrhythmias induced when exogenous catecholamines are
given.Y6-38 This difference in efficacy of antiarrhythmic
activity may indicate that suppression of endogenous
norepinephrine release is an additional mechanism for the
potent antiarrhythmic property of class I agents in ischemia. In addition, although clinical studies have shown
that 3-blockers without membrane stabilizing activity reduce sudden cardiac death in patients after acute myocardial infarction,' it is not known if they are as effective as
those with such activity in the inhibition of ventricular
arrhythmias during acute myocardial ischemia.
The generally accepted concept that the membrane
stabilizing activity of ,3-antagonists is of little therapeutic importance is based primarily on data collected
under physiological conditions. By simulating the metabolic changes seen in acute myocardial ischemia, we
have shown a suppression of norepinephrine release by
propranolol at concentrations found clinically.39-41 The
importance of the experimental environment in the
assessment of drug effects in cardiovascular tissues has
been stressed by previous studies.9-6,28 The present
study, concerning neural norepinephrine release during
myocardial ischemia, provides another example of this
1892
Circulation Vol 88, No 4, Part 1 October 1993
issue whereby ischemic changes amplify drug effects
leading to unexpected pharmacological actions.
Acknowledgments
The Cardiovascular Research Unit is supported by the
British Heart Foundation. The part of this project conducted
in Baker Medical Research Institute was supported by grants
from the Alfred Group of Hospitals, Melbourne, and BP
Australia Ltd. The excellent technical assistance of Miss
Margaret Millar and Mrs Jean Samuel is greatly acknowledged. We thank Dr A. Ungar for his generous supply of
butoxamine. We are grateful to Professor James Angus and
Professor Murray Esler for their support with laboratory
facilities. We also wish to thank Miss Helen Cox and Miss
Andrea Turner for their help in the catechol assay.
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extracellular potassium and ischemia.
X J Du, R A Riemersma, K A Fox and A M Dart
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Circulation. 1993;88:1885-1892
doi: 10.1161/01.CIR.88.4.1885
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