| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© 1996 American Heart Association, Inc.
Articles |
Transient Ischemia Reduces Norepinephrine Release During Sustained Ischemia
Neural Preconditioning in Isolated Rat Heart
Presented in part at the 67th Scientific Sessions of the American Heart Association, Dallas, Tex, November 14-17, 1994.
From I. Medizinische Klinik, Technische Universität München (Germany).
Correspondence to Dr M. Seyfarth, I. Medizinische Klinik der Technischen Universität München, Klinikum rechts der Isar, Ismaninger Str. 22, 81675 München, Germany. E-mail seyfarth@med1.med.tu-muenchen.de.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and Methods Results Discussion References |
|---|
Abstract Endogenous catecholamine release may play a role in ischemic preconditioning either as a trigger or as a target within the process of myocardial preconditioning. Therefore, we investigated the effect of transient ischemia (TI) on norepinephrine release during sustained ischemia in isolated rat hearts. TI was induced by multiple cycles of global ischemia followed by reperfusion with a duration of 5 minutes each, comparable to ischemic preconditioning protocols. After TI, norepinephrine release was evoked by either sustained global ischemia, anoxia, cyanide intoxication, tyramine, or electrical stimulation. During TI, no washout of norepinephrine was observed, and tissue concentrations of norepinephrine were not changed. TI, however, reduced norepinephrine overflow after 20 minutes of sustained ischemia from 239±26 pmol/g (control) to 79±8 pmol/g (67% reduction, P<.01). A similar reduction of ischemia-induced norepinephrine release from 192±22 pmol/g (control) to 90±15 pmol/g was observed when hearts underwent transient anoxia without glucose (P<.05). When reperfusion between TI and sustained ischemia was prolonged from 5 to 90 minutes, the inhibitory effect of TI on norepinephrine release was gradually lost. Susceptibility to TI was a unique feature of norepinephrine release induced by sustained ischemia, since release of norepinephrine evoked by anoxia, cyanide intoxication, tyramine, or electrical stimulation remained unaffected by TI. We propose a protective effect of TI on neural tissue, which may reduce norepinephrine-induced damage during prolonged myocardial ischemia.
Key Words: norepinephrine release • preconditioning • myocardial ischemia • rat hearts • anoxia
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and Methods Results Discussion References |
|---|
Ischemic preconditioning reduces the extent of myocardial injury in a subsequent myocardial infarction in most species, including dogs,1 pigs,2 rabbits,3 and rats.4 There is also evidence that ischemic preconditioning occurs in human myocardium.5 6 So far, the mechanism underlying the protective effects of preconditioning remains uncertain. Some authors suggest a role of catecholamines and adrenergic receptors in the reduction of infarct size by preconditioning.7 8 9 10 Additionally, ischemic preconditioning can reduce the occurrence of ischemia-related arrhythmias, probably by a pathway different from the pathway mediating the anti-infarct effects of preconditioning.11 Suppression of arrhythmias could be caused by antiadrenergic effects,12 potentially induced by transient ischemia.
Generally, during sustained myocardial ischemia, norepinephrine accumulation within ischemic myocardium is mainly caused by a locally induced nonexocytotic release of norepinephrine.13 14 Recent findings indicate that nonexocytotic release is also the underlying mechanism of ischemia-evoked norepinephrine release in human myocardium.15 Nonexocytotic release of norepinephrine during ischemia, which is a consequence of energy starvation of the sympathetic nerve terminal, contributes to the genesis of malignant arrhythmias12 16 and accelerates the development of myocardial injury17 in experimental ischemia. Thus, it would be important to determine whether transient ischemia can protect neural tissue as it does myocardial tissue. The present study, therefore, investigated the effect of transient ischemia on norepinephrine release during a sustained ischemic period. The experiments were performed in isolated perfused rat hearts to exclude systemic influences and to ensure that findings are caused by factors inherent to the heart. This model has been established in previous studies of norepinephrine release during myocardial ischemia.13 14 18
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and Methods Results Discussion References |
|---|
Male Wistar rats (200 to 250 g, Thomae, Biberach, Germany) were anesthetized with thiopental sodium (75 to 100 mg/kg IP). After injection of 500 U heparin intravenously, the thorax was opened. Hearts were excised into 4°C Krebs-Henseleit solution and weighed (mean weight, 0.85±0.03 g). The aorta was cannulated for isolated perfusion (Langendorff technique) within 30 seconds after excision, as has been described previously.14 Hearts were perfused at a constant flow of either 6 or 12 mL/min per gram heart weight with a modified Krebs-Henseleit solution (mmol/L: NaCl 125, NaHCO3 16.9, Na2HPO4 0.2, KCl 4.0, CaCl2 1.85, MgCl2 1.0, glucose 11.0, and sodium EDTA 0.027) and gassed with 95% O2/5% CO2, achieving a Po2 of
300 mm Hg, a
Pco2 of 35 to 45 mm Hg, and a pH of 7.35 to 7.45.
Perfusion pressure was monitored in some series of experiments to
calculate postischemic increase of perfusion pressure in
hearts with and without preceding transient ischemia (Statham
pressure transducer). The temperature of the hearts and
perfusate was kept constant at 37.5°C.
Experimental Design
Experiments were performed with 6 to 12
hearts at the same time,
divided into two groups with and without transient ischemia
(each group, three to six hearts). After 20 to 30 minutes of
equilibration, the group with transient ischemia was treated
with a standard transient ischemia/reperfusion protocol
including one to four 5-minute periods of global stop-flow
ischemia, each followed by 5 minutes of reperfusion. The
subsequent sustained ischemia (20 or 40 minutes) was carried
out in both groups. To assess reversibility of the effect induced by
transient ischemia, the reperfusion time immediately before the
sustained ischemia of 20 minutes was extended to 20, 40, or 90
minutes in additional experiments. Furthermore, we investigated the
effect of transient anoxia on ischemia-induced
norepinephrine release. One group was treated with three
periods of 3-minute anoxia, each followed by 5 minutes of normoxic
reperfusion. Anoxia was induced by perfusing the hearts with a
glucose-free solution gassed with 95% N2/5%
CO2 at an unchanged flow rate.18 A
Po2 of <5 mm Hg was achieved within 1 minute after the
addition of 1.0 mmol/L sodium dithionite (Merck). Subsequently, all
hearts were subjected to the standard 20 minutes of
ischemia.
In separate sets of experiments, norepinephrine release was induced by stimuli, different from ischemia, to investigate the effect of transient ischemia on these mechanisms of norepinephrine release. Therefore, hearts pretreated with transient ischemia (three periods of 5-minute ischemia, each followed by reperfusion of 5 minutes) and respective control hearts were subsequently subjected to either an anoxic perfusion or a perfusion with cyanide (1 mmol/L, Merck) for 50 minutes each to block oxidative phosphorylation.18 Anoxia was induced in the same manner as described above. Additionally, the effect of transient ischemia on norepinephrine release induced by tyramine (100 µmol/L for 40 minutes, Sigma) was tested. Finally, we investigated the effect of transient ischemia on exocytotic norepinephrine release evoked by electrical field stimulation. Electrical field stimulation (1 minute, 5 V, 6 Hz) in this model has been described previously.13 After transient ischemia (three periods of 5-minute ischemia, each followed by reperfusion of 5 minutes), electrical stimulation was performed to induce norepinephrine release. The effect of transient ischemia on stimulation-evoked norepinephrine release was evaluated by comparing release in hearts with and without transient ischemia.
A further series of
experiments was performed to investigate the effect
of activation or blockade of receptors potentially involved in the
mechanism of ischemic preconditioning on
ischemia-induced norepinephrine release.
Adenosine receptors were activated by adenosine
(100 µmol/L, Sigma); 
1-receptors, by
phenylephrine (10 µmol/L, Sigma). Both agonists were
added to the perfusate after the 20-minute equilibration period
three times for 5 minutes each, parallel to the protocol with transient
ischemia. Subsequently, 20 minutes of global ischemia
was induced. In a second experiment, hearts were divided into three
groups. The first group served as a control without transient
ischemia. In the second and third groups, transient
ischemia was induced (three periods of 5-minute global
ischemia), with and without infusion of receptor
antagonists (10 µmol/L 8-phenyltheophylline and 1
µmol/L prazosin, Sigma) or inhibitors of protein kinase C
(0.1 µmol/L bisindolylmaleimide HCl, Calbiochem) throughout the
experiment, starting 10 minutes before the onset of the first transient
ischemia. All hearts were subjected to a final ischemic
period of 20 minutes.
Determination of Norepinephrine
Samples for endogenous
norepinephrine
release were consecutively taken from the effluent for 1 minute. In a
separate set of experiments, hearts were perfused at 12 mL/min per gram
heart weight to investigate how changes of coronary flow
interfere with washout kinetics and the effect of transient
ischemia on ischemia-induced
norepinephrine release. In these experiments, the
collection time of each sample was 15 seconds during the first minute
of reperfusion and 30 seconds during the second to sixth minute of
reperfusion to determine washout kinetics during reperfusion. All
samples were cooled on ice, stabilized by the addition of
Na2EDTA (10 mmol/L), and stored at -60°C until
assayed. In a separate experiment, the effect of transient
ischemia on the tissue content of norepinephrine
and its metabolite dihydroxyphenyl glycol (DOPEG) was determined by
measuring tissue concentration after transient ischemia. Hearts
were frozen in liquid nitrogen immediately after the third cycle of
transient ischemia and reperfusion. Thereafter, hearts were
homogenized, and norepinephrine and DOPEG were
determined in the liquid phase.18 For comparison, the
norepinephrine content of hearts without transient
ischemia and the norepinephrine content of freshly
isolated hearts were measured.
Norepinephrine and DOPEG were measured by using a high-performance liquid chromatography (HPLC) method.14 18 Briefly, after a two-step extraction, separation was performed with a reversed-phase counter-ion HPLC system. Electrochemical detection was used for quantitative analysis. Recovery was 98%, and the limit of detection was 0.1 nmol/L.
Calculation and Statistics
Norepinephrine release induced by
ischemia
was calculated as cumulative norepinephrine overflow
obtained within 5 minutes of washout after the sustained
ischemic period. Norepinephrine release induced by
anoxia, cyanide intoxication, or tyramine infusion was measured every
fifth minute during these interventions and was calculated by linear
interpolation and integration.18
Norepinephrine release induced by electrical stimulation
was determined during 1 minute of electrical stimulation and during the
following 2 minutes. Results are given as arithmetic mean±SEM.
Statistical differences were tested either by unpaired Student's
t test for two groups or by multicomparison ANOVA followed
by Scheffé's F test (P<.05).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and Methods Results Discussion References |
|---|
Norepinephrine Release Induced by Sustained Ischemia
Norepinephrine concentrations were below the detection limit (0.1 nmol/L) in all samples collected immediately after the 5-minute transient ischemic periods. In order to exclude undetectable loss of norepinephrine or degradation of catecholamines during transient ischemia, tissue concentrations of norepinephrine and its metabolite DOPEG were determined at the end of transient ischemia in a separate experiment. Three cycles of 5-minute transient ischemia, each followed by 5 minutes of reperfusion, affected neither the tissue concentration of norepinephrine (control hearts without transient ischemia, 4418±126 pmol/g; hearts with transient ischemia, 4334±136 pmol/g; each group, n=12) nor the tissue concentration of DOPEG (<100 pmol/g for each group). Additionally, isolation and perfusion of hearts did not alter norepinephrine content, since norepinephrine concentration of hearts, frozen immediately after isolation, was not significantly different (4669±334 pmol/g, n=6).
Without
transient ischemia, sustained ischemia for 20
minutes induced an endogenous release of
norepinephrine, which amounted to 239±26 pmol/g (n=17).
One cycle of 5-minute transient ischemia followed by 5 minutes
of reperfusion reduced ischemia-induced
norepinephrine release to 107±17 pmol/g (55% reduction,
n=11); two cycles resulted in a release of 71±6 pmol/g (70%
reduction, n=13). Three and four cycles did not further reduce
ischemia-induced norepinephrine release (79±8
pmol/g [67% reduction, n=26] and 102±21 pmol/g
[57% reduction,
n=10], respectively). Norepinephrine release in all groups
with transient ischemia was statistically different from the
release in the group without transient ischemia
(P<.01) (Fig 1
). The inhibitory
effect by transient ischemia was reproduced in experiments with
a flow of 12 mL/min per gram heart weight. Three and four cycles of
5-minute transient ischemia reduced
ischemia-induced norepinephrine release from
258±26 pmol/g (control, n=18) to 90±22 pmol/g (65%
reduction) and
125±20 pmol/g (52% reduction), respectively (each group, n=8;
P<.05) (Fig 1).
|
The washout kinetics of
norepinephrine during reperfusion
is demonstrated in the top panel of Fig 2. The time
course of norepinephrine release is best described by a
two-compartment model in hearts both with and without transient
ischemia (Fig 2, bottom panel). Regression analysis
shows a half-time of 12.6 seconds (11.0 seconds in hearts with
transient ischemia) for washout from the first compartment and
107 seconds (104 seconds in hearts with transient ischemia) for
washout from the second compartment. This is based on the following
calculated curve formulas:
c(t)=58·exp(-3.3·t)+2.5· exp(-0.39·t)
in
control hearts and
c(t)=37·exp(-3.8·t)+1.2·exp(-0.40·t)
in
hearts with transient ischemia (R2>.98
for all regression curves). Washout from both compartments was
comparably reduced by transient ischemia (first compartment,
45%; second compartment, 53%). In both groups, nearly 80% of
norepinephrine release is determined by washout from the
first compartment.
|
The inhibitory effect of three cycles of transient
ischemia was gradually lost when the reperfusion period before
the sustained ischemia was prolonged to 20, 40, and 90 minutes
(Fig 3, top panel). Stepwise norepinephrine
release reached control values when the reperfusion time was prolonged
from 5 minutes (67% reduction) to 20 minutes (26%), 40 minutes
(20%), and 90 minutes (13% reduction), thereby demonstrating
reversibility of the effect by transient ischemia. When the
sustained ischemic period was prolonged to 40 minutes, three
cycles of 5-minute transient ischemia still resulted in a
small, but statistically not significant, reduction of
norepinephrine release (506±45 versus 659±67 pmol/g; each
group, n=12; P=.07) (Fig 3, bottom
panel).
|
Fig 4 demonstrates the increase of perfusion
pressure
during reperfusion after 20 minutes of sustained ischemia in
hearts both with and without transient ischemia (three cycles
of 5-minute transient ischemia) compared with the initial
perfusion pressure after the equilibration period. The mean initial
perfusion pressure was 50±3 mm Hg in control hearts (n=15) and
58±3
mm Hg in hearts randomized for subsequent transient ischemia
(n=16). Perfusion pressure immediately before 20 minutes of sustained
ischemia did not differ between both groups (53±3 mm Hg in
control hearts and 51±2 mm Hg in hearts with transient
ischemia). The postischemic increase of perfusion
pressure was significantly lower in hearts with transient
ischemia up to 10 minutes of reperfusion.
|
Transient Ischemia Versus Transient Anoxia
Three cycles of
3-minute anoxic and glucose-free perfusion
each followed by 5-minute normoxic perfusion resulted in a reduction of
norepinephrine release induced by 20 minutes of sustained
ischemia from 192±22 pmol/g (n=13) to 90±15 pmol/g (53%
reduction, n=8, P<.05) (Fig 5). Conversely,
transient anoxic perfusion in the presence of glucose did not affect
the ischemia-induced release of norepinephrine
(190±29 pmol/g, n=6). This demonstrates that attenuation of
ischemia-induced norepinephrine release depends
on complete energy starvation, either by global ischemia or
anoxia in combination with a lack of glucose. During transient anoxic
and glucose-free perfusion, no overflow of
norepinephrine was detected from any of the hearts.
|
Involvement of Receptors in Mediating Neural
Preconditioning
To test the hypothesis that reduction of
ischemia-induced norepinephrine release is
mediated by activation of adenosine or
1-receptors during transient ischemia, the
effect of receptor activation and receptor blockade on
ischemia-induced norepinephrine release was
studied (Fig 6). Neither the administration of
adenosine (100 µmol/L) nor of phenylephrine (10
µmol/L) as different "preconditioning stimuli" reduced
ischemia-induced norepinephrine release
(181±39 pmol/g [adenosine] versus 184±45 pmol/g
[corresponding control value]; 271±36 pmol/g
[phenylephrine] versus 280±39 [corresponding control
value]; each group, n=6). Furthermore, blockade of these receptors
by
8-phenyltheophylline (10 µmol/L) and prazosin (1 µmol/L) did not
abolish the effect of transient ischemia on
ischemia-induced norepinephrine release (hearts
with 8-phenyltheophylline, 78% reduction; hearts without
8-phenyltheophylline, 53% reduction; hearts with prazosin, 82%
reduction; hearts without prazosin, 72% reduction; each group, n=8).
8-Phenyltheophylline and prazosin per se had no effect on
ischemia-induced norepinephrine release.
Additionally, the protecting effect of transient ischemia was
only partially abolished by inhibition of protein kinase C
(bisindolylmaleimide, 0.1 µmol/L), since norepinephrine
release in hearts with transient ischemia and protein kinase C
inhibition was still significantly reduced (49% reduction, n=8,
P<.05). In comparison, the effect of transient
ischemia without protein kinase C inhibition resulted in 71%
reduction of norepinephrine release (n=8, P<.05) in this
experiment (Fig 6). Protein kinase C inhibition per se did not
significantly change ischemia-induced
norepinephrine release.
|
Norepinephrine Release Induced by Anoxia and
Cyanide Intoxication
To characterize the protective effects of
transient
ischemia on neural tissue, we further examined the effect of
transient ischemia on norepinephrine release
induced by sustained energy depletion in contrast to stop-flow
ischemia. Sustained energy depletion with continued perfusion
was caused either by anoxic perfusion or by cyanide intoxication, both
combined with glucose-free perfusion. Without transient
ischemia (three cycles of 5-minute ischemia), sustained
anoxia induced an overflow of norepinephrine starting after
5 minutes and reaching a peak value (136 pmol/g per minute) after 20
minutes (Fig 7, top panel). The total amount of
norepinephrine induced by 50 minutes of anoxia was 3145±84
pmol/g (n=6). Three cycles of 5-minute transient ischemia given
before anoxic perfusion did not affect time course, peak value, or
total amount (3087±138 pmol per gram, n=6) of the release
induced by
anoxia (Fig 7, top panel).
|
The investigation of cyanide
intoxication yielded corresponding results
(Fig 7
, bottom panel). Onset and peak value of
norepinephrine release induced by cyanide intoxication were
not different between hearts with and without transient
ischemia. The cumulative overflow of norepinephrine
after 50 minutes of cyanide intoxication was 1643±79 pmol/g in hearts
with transient ischemia versus 1770±111 pmol/g in control
hearts (each group, n=6). During cyanide intoxication, axoplasmic
norepinephrine is partially deaminated to its metabolite
DOPEG, which diffuses passively to the extracellular
space.18 Like norepinephrine, there was no
difference in time course and cumulative overflow of DOPEG (hearts with
transient ischemia, 616±55 pmol/g; control hearts, 541±38
pmol/g; each group, n=6) between both groups.
Norepinephrine Release Induced by Tyramine or
Electrical Stimulation
Finally, we investigated the effect of
transient ischemia
on norepinephrine release caused by indirectly acting
sympathomimetic amines, such as tyramine, or by stimuli of exocytotic
release, such as electrical field stimulation. As shown in Fig
8, top panel, three cycles of 5-minute transient
ischemia did not change the release of
norepinephrine induced by tyramine. The cumulative overflow
after 40 minutes was 1460±137 pmol/g in hearts with transient
ischemia versus 1482±137 pmol/g in control hearts (each group,
n=6).
|
Likewise, transient ischemia did not affect the amount of
exocytotic norepinephrine release evoked by electrical
field stimulation. Electrical stimulation, which was performed during
the fifth minute of reperfusion after three cycles of 5-minute
transient ischemia, caused a release of 92±9 pmol/g
norepinephrine in control hearts and of 107±17 pmol/g
norepinephrine in hearts with transient ischemia
(each group, n=7) (Fig 8, bottom panel).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and Methods Results Discussion References |
|---|
Previous studies demonstrated a reduction of infarct size4 19 and postischemic myocardial dysfunction,9 20 as well as a reduction of both ischemia-induced21 22 and reperfusion-induced21 23 24 malignant arrhythmias by ischemic preconditioning in rat hearts. The principal finding of the present study was a protective effect of transient ischemia on neural tissue during ischemia in rat hearts. Nonexocytotic norepinephrine release during ischemia was suppressed by transient ischemia in a manner comparable to the reduction in infarct size and ischemia-induced arrhythmias by ischemic preconditioning.
Effect of Transient Ischemia on Norepinephrine
Release Induced by Ischemia
Although a single 5-minute ischemic
episode was sufficient
to reduce ischemia-induced norepinephrine
release, the most pronounced effect of transient ischemia, with
a reduction of norepinephrine release to 30% of control,
was achieved with two or three ischemia/reperfusion cycles.
This suppression could not be further enhanced by an additional cycle
of transient ischemia. Since there was no statistical
difference between one and four cycles of transient ischemia,
an "all-or-nothing" effect on
ischemia-induced norepinephrine release cannot
be excluded. Previous studies in rat hearts have demonstrated that one
preconditioning cycle is sufficient to reduce infarct size and
arrhythmias but that the strongest effect was achieved by
multiple cycles.4 19 25 The protective
effects of short
preceding ischemic episodes on norepinephrine
release were reversible by prolonging the interval to the final
ischemia, corresponding to prior findings by Li et
al,22 who also documented a transitory effect of
ischemic preconditioning on myocardial parameters.
Hence, time course, extent, and reversibility of the observed
attenuation of ischemia-induced norepinephrine
release resembles the protective effect of preconditioning on
previously studied end points of ischemia, such as infarct size
and ischemia-related arrhythmias in rat hearts.
It has been reported that hypoxic preconditioning has a protective effect on infarct size in dogs that is comparable to ischemic preconditioning.26 Likewise, transient anoxic perfusion reduced ischemia-induced norepinephrine release, as long as anoxia was combined with glucose-free perfusion. Therefore, the mechanism underlying the effect of transient ischemia on norepinephrine release during a sustained ischemic period depended on a complete blockade of ATP production from both oxidative phosphorylation and anaerobic glycolysis. A rapid fall of ATP in neurons during energy depletion has been demonstrated, since cytosolic ATP of adrenal chromaffin cells declines with a half-life of 5 minutes during metabolic inhibition with cyanide.27 Furthermore, because flow is preserved during anoxia, the extracellular accumulation of metabolites during transient energy depletion appears not to be mandatory for initiating a protective effect.
Within the cycles of transient ischemia
and anoxia,
norepinephrine overflow was not observed. A delayed onset
of ischemia-evoked norepinephrine release is
consistent with previous investigations, which defined the
detectable start of nonexocytotic norepinephrine release at
10 minutes of global and normothermic ischemia
in rat hearts.14 18 Additionally, transient
ischemic episodes did not affect tissue stores of
norepinephrine and DOPEG in our experiments. This
observation excluded the possibility that the reduction of
norepinephrine release during a sustained ischemic
period was caused by release or degradation of
catecholamines before the sustained ischemic
period. The reversibility of the effect of transient ischemia
on norepinephrine release further supports the view that
functional rather than structural (ie, depletion of
norepinephrine) changes were responsible for the observed
protection of neural tissue.
Ischemic preconditioning attenuates postischemic contracture, thereby reducing extravascular compressive forces.20 Accordingly, we could observe a reduced increase of perfusion pressure during reperfusion after sustained ischemia in hearts with preceding transient ischemia. Whereas the attenuation of postischemic contracture can serve as an indicator of myocardial protection in a model of global ischemia, it could also affect the washout of norepinephrine during reperfusion. However, in both groups, half-times of norepinephrine washout kinetics did not differ, and 80% of the total norepinephrine overflow after ischemia came from a first compartment, presumably from the interstitial space, where norepinephrine accumulates during ischemia. About 20% of norepinephrine overflow was slowly washed out from a second compartment, in keeping with a neuronal release during reperfusion. Additionally, a flow-dependent effect could be excluded, since transient ischemia attenuated ischemia-induced norepinephrine release in a similar manner in experiments with flow rates of both 6 and 12 mL/min per gram heart weight.
Effect of Transient Ischemia on Norepinephrine
Release Induced by Stimuli Different From
Ischemia
We expected insights into the underlying mechanism of neural
preconditioning from experiments with norepinephrine
release evoked by sustained anoxia and cyanide intoxication, which were
used as different means to induce energy starvation.
Norepinephrine release induced by either anoxia or cyanide
intoxication, however, was not affected by transient ischemia.
This was true for time course and total amount of
norepinephrine overflow during the period of energy
depletion. Likewise, during the experiments with cyanide intoxication,
the overflow of DOPEG remained unaffected by transient
ischemia, indicating that axoplasmic norepinephrine
concentration remained unchanged after transient
ischemia.18 Nevertheless, the lack of effect of
transient ischemia on norepinephrine release
induced by anoxia and cyanide intoxication may help to create
hypotheses about the mechanism of neural preconditioning. Since the
accumulation of metabolites28 29 and ionic
alterations20 30 31 are more pronounced
in
ischemia compared with anoxia or cyanide intoxication when flow
is preserved, the latter disturbances might be a target of
transient ischemia. It has been shown that an activated
Na+-H+ exchange in neurons is mandatory for
ischemia-induced norepinephrine release but not
for norepinephrine release induced by cyanide
intoxication.32 A specific reduction of
ischemia-induced norepinephrine release could
therefore be explained by a diminished activation of
Na+-H+ exchange after transient
ischemia.
Transient ischemia did not modulate norepinephrine release caused by tyramine, an indirectly acting amine, thereby excluding an interaction with the mechanism of tyramine-induced release.33 Likewise, exocytotic norepinephrine release evoked by electrical field stimulation was unaffected by transient ischemia. This corresponds with previous findings in our model, documenting a full recovery of stimulation-evoked neurotransmitter release after brief ischemic periods.34 Accordingly, Miyazaki and Zipes35 described a preserved autonomic function during reperfusion after ischemic preconditioning. Thus, susceptibility to transient ischemia was a unique feature of norepinephrine release during ischemia.
Implications of Reduced Ischemia-Induced
Norepinephrine Release for the Preconditioning Effect of
Myocardial Tissue
An involvement of adrenergic stimulation in ischemic
preconditioning, so far, has been attributed to a release of
norepinephrine during ischemic preconditioning that
may activate the myocytes via 1-receptors and
protein kinase C, thereby leading to beneficial effects in a subsequent
sustained ischemic period.9 10 However, the
mandatory role of catecholamines in ischemic
preconditioning in terms of reduced infarct size is
controversial.7 36 37 Our results do not
document a
detectable release of norepinephrine during transient
ischemia (at least in isolated perfused rat hearts). The data
rather suggest that sympathetic nerve terminals themselves are involved
in adaptation to ischemia in a manner comparable to that of
myocardial tissue. This means that protection of ischemic
hearts by preconditioning is reflected not only by reduced infarct size
and less severe arrhythmias but also by the suppression of
ischemia-induced norepinephrine release. Since
interstitial accumulation of norepinephrine
accelerates the damage of ischemic
myocardium17 38 and, particularly, contributes
to ischemia-induced
arrhythmias,16 39 40 41
inhibition of nonexocytotic
norepinephrine release may postpone or attenuate
ischemic injury of the myocardium. The lack of
receptor activation by adenosine or phenylephrine
to mimic neural preconditioning and the lack of receptor blockade to
abolish the effect of transient ischemia on
ischemia-induced norepinephrine release suggest
that neural preconditioning is mediated differently from the
preconditioning of myocardial tissue. This assumption is further
substantiated by the independence of neural preconditioning from
protein kinase C activation, since bisindolylmaleimide, a protein
kinase C inhibitor,42 did not abolish the
effect of transient ischemia on ischemia-induced
norepinephrine release. Activation of protein kinase C
seems to be mandatory for reduction of infarct size by ischemic
preconditioning.11 43 44 Therefore, an
association between
the reduction of ischemia-induced
norepinephrine release and infarct size is unlikely. There
is, however, evidence reported in a recent study12 that
ischemia-induced norepinephrine release is
closely related to the incidence of ischemia-induced
arrhythmias in rat hearts, since pharmacological suppression of
norepinephrine release abolished the incidence of
ventricular arrhythmias during ischemia.
The protection of ischemic myocardium in terms of
reduced arrhythmias may therefore be explained by the reduction
of ischemia-induced norepinephrine release in
rat hearts. Accordingly, previous studies have suggested that different
signaling pathways could be responsible for a concomitant protective
effect of transient ischemia on arrhythmias and infarct
size.11
|
Acknowledgments |
|---|
This study was supported by a grant from the Deutsche Forschungsgemeinschaft (Ri 423/2-1). We would like to thank Maike Lücking and Sonja Fendt for skillful preparation and technical assistance.
Received July 12, 1995; accepted December 14, 1995.
|
References |
|---|
|
TopAbstractIntroductionMaterials and Methods Results Discussion References |
|---|
1. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation. 1986;74:1124-1136.
2.
Schott RJ, Rohmann S, Braun ER, Schaper W.
Ischemic preconditioning reduces infarct size in swine
myocardium. Circ Res. 1990;66:1133-1142.
3.
Cohen MV, Liu GS, Downey JM. Preconditioning
causes improved wall motion as well as smaller infarcts after transient
coronary occlusion in rabbits.
Circulation. 1991;84:341-349.
4.
Liu Y, Downey JM. Ischemic
preconditioning protects against infarction in rat heart.
Am J Physiol. 1992;263:H1107-H1112.
5.
Deutsch E, Berger M, Kussmaul WG, Hirshfeld JW Jr,
Gerrmann HC, Laskey WK. Adaptation to ischemia during
percutaneous transluminal coronary angioplasty:
clinical, hemodynamic and metabolic
features. Circulation. 1990;82:2044-2051.
6. Yellon DM, Alkhulaifi AM, Pugsley WB. Preconditioning the human myocardium. Lancet. 1993;342:276-277. [Medline] [Order article via Infotrieve]
7.
Toombs CF, Wiltse AL, Shebuski RJ.
Ischemic preconditioning fails to limit infarct size in
reserpinized rabbit myocardium: implication of
norepinephrine release in the preconditioning
effect. Circulation. 1993;88:2351-2358.
8.
Thornton JD, Daly JF, Cohen MV, Yang X-M, Downey
JM. Catecholamines can induce adenosine
receptor–mediated protection of the myocardium but do
not participate in ischemic preconditioning in the
rabbit. Circ Res. 1993;73:649-655.
9.
Banerjee A, Locke-Winter C, Rogers KB, Mitchell MB,
Brew EC, Cairns CB, Bensard DD, Harken AH. Preconditioning
against myocardial dysfunction after ischemia and
reperfusion by an 1-adrenergic mechanism.
Circ Res. 1993;73:656-670.
10.
Bankwala Z, Hale SL, Kloner RA. -Adrenoceptor
stimulation with exogenous norepinephrine or release of
endogenous catecholamines mimics
ischemic preconditioning.
Circulation. 1994;90:1023-1028.
11.
Speechly-Dick ME, Mocanu MM, Yellon DM. Protein
kinase C: its role in ischemic preconditioning in the
rat. Circ Res. 1994;75:586-590.
12. Kurz T, Offner B, Schreieck J, Richardt G, Tölg R, Schömig A. Nonexocytotic noradrenaline release and ventricular fibrillation in ischemic rat hearts. Naunyn Schmiedebergs Arch Pharmacol. 1995;352:491-496. [Medline] [Order article via Infotrieve]
13.
Seyfarth M, Feng Y, Hagl S, Sebening F, Richardt G,
Schömig A. Effect of myocardial ischemia on
stimulation-evoked noradrenaline release: modulated
neurotransmission in rat, guinea pig, and human cardiac tissue.
Circ Res. 1993;73:496-502.
14.
Schömig A, Dart AM, Dietz R, Mayer E,
Kübler W. Release of endogenous
catecholamines in the ischemic
myocardium of the rat, A: locally mediated release.
Circ Res. 1984;55:689-701.
15. Kurz T, Richardt G, Hagl S, Seyfarth M, Schömig A. Two different mechanisms of noradrenaline release during normoxia and simulated ischemia in human cardiac tissue. J Mol Cell Cardiol. 1995;27:1161-1172. [Medline] [Order article via Infotrieve]
16.
Corr PB, Gillis RA. Autonomic neural influences
on the dysrhythmias resulting from myocardial infarction.
Circ Res. 1978;43:1-9.
17. Rona G. Catecholamine cardiotoxicity. J Mol Cell Cardiol. 1985;17:291-306. [Medline] [Order article via Infotrieve]
18.
Schömig A, Fischer S, Kurz T, Richardt G,
Schömig E. Nonexocytotic release of endogenous
noradrenaline in the ischemic and anoxic rat heart:
mechanism and metabolic requirements.
Circ Res. 1987;60:194-205.
19.
Yellon DM, Alkhulaifi AM, Browne EE, Pugsley WB.
Ischemic preconditioning limits infarct size in the rat
heart. Cardiovasc Res. 1992;26:983-987.
20.
Asimakis GK, Inners-McBride K, Medellin G, Conti
VR. Ischemic preconditioning attenuates acidosis and
postischemic dysfunction in isolated rat heart.
Am J Physiol. 1992;263:H887-H894.
21. Vegh A, Komoro S, Szekeres L, Parratt JR. Antiarrhythmic effects of preconditioning in anaesthetised dogs and rats. Cardiovasc Res. 1992;26:487-495. [Medline] [Order article via Infotrieve]
22. Li Y, Whittaker P, Kloner RA. The transient nature of the effect of ischemic preconditioning on myocardial infarct size and ventricular arrhythmia. Am Heart J. 1992;123:346-353. [Medline] [Order article via Infotrieve]
23.
Shiki K, Hearse DJ. Preconditioning of
ischemic myocardium: reperfusion-induced
arrhythmias. Am J Physiol. 1987;253:H1470-H1476.
24.
Hagar JM, Hale SL, Kloner RA. Effects of
preconditioning ischemia on reperfusion arrhythmias
after coronary artery occlusion and reperfusion in the
rat. Circ Res. 1991;68:61-68.
25.
Li GC, Vasquez BS, Gallagher KP, Lucchesi BR.
Myocardial protection with preconditioning.
Circulation. 1990;82:609-619.
26. Shizukuda Y, Mallet RT, Lee SC, Downey HF. Hypoxic preconditioning of ischaemic canine myocardium. Cardiovasc Res. 1992;26:534-542. [Medline] [Order article via Infotrieve]
27. Corcoran JJ, Korner M, Caughey B, Kirshner N. Metabolic pools of ATP in cultured bovine adrenal medullary chromaffin cells. J Neurochem. 1986;47:945-952. [Medline] [Order article via Infotrieve]
28. Belardinelli L, Linden J, Berne R. The cardiac effects of adenosine. Prog Cardiovasc Dis. 1989;32:73-97. [Medline] [Order article via Infotrieve]
29.
Downey JM, Liu GS, Thornton JD.
Adenosine and the anti-infarct effects of
preconditioning. Cardiovasc Res. 1993;27:3-8.
30.
Steenbergen C, Perlman ME, London RE, Murphy E.
Mechanism of preconditioning: ionic alterations.
Circ Res. 1993;72:112-125.
31.
de Albuquerque CP, Gerstenblith G, Weiss RG.
Importance of metabolic inhibition and cellular pH in
mediating preconditioning contractile and metabolic effects
in rat hearts. Circ Res. 1994;74:139-150.
32.
Schömig A, Kurz T, Richardt G, Schömig
E. Neuronal sodium homoeostasis and axoplasmic amine
concentration determine calcium-independent
noradrenaline release in normoxic and ischemic rat
heart. Circ Res. 1988;63:214-226.
33. Bönisch H, Trendelenburg U. The mechanism of action of indirectly acting sympathomimetic amines. In: Trendelenburg U, Weiner N, eds. Catecholamines I (Handbook Experimental Pharmacology 90/I). Berlin, Germany: Springer-Verlag; 1988:247-277.
34. Seyfarth M, Feng Y, Kranzhöfer R, Richardt G, Schömig A. Recovery of stimulation-evoked noradrenaline release during reperfusion after acute ischemia in rat hearts. J Mol Cell Cardiol. 1992;24(suppl V):17. Abstract.
35.
Miyazaki T, Zipes DP. Protection against
autonomic denervation following acute myocardial infarction by
preconditioning ischemia. Circ
Res. 1989;64:437-448.
36. Ardell JL, Yang XM, Thornton JD, Swafford A, Cohen MV, Downey JM. Depletion of norepinephrine by chronic surgical sympathectomy does not block protection from ischemic preconditioning. Circulation. 1994;90(suppl I):I-108. Abstract.
37.
Tsuchida A, Liu Y, Liu GS, Cohen MV, Downey
JM. 1-Adrenergic agonists precondition
rabbit ischemic myocardium independent of
adenosine by direct activation of protein kinase C.
Circ Res. 1994;75:576-585.
38. Waldenström AP, Hjalmarson AC, Thornell L. A possible role of noradrenaline in the development of myocardial infarction. Am Heart J. 1978;95:43-51. [Medline] [Order article via Infotrieve]
39. Dietz R, Offner B, Dart AM, Schömig A. Ischemia-induced noradrenaline release mediates ventricular arrhythmias. In: Brachmann J, Schömig A, eds. Adrenergic System and Ventricular Arrhythmias in Myocardial Infarction. Berlin, Germany: Springer-Verlag; 1989:313-321.
40.
Ebert PA, Vanderbeck RB, Allgood RJ, Sabiston DC Jr.
Effect of chronic cardiac denervation on arrhythmias of
myocardial infarction. Cardiovasc Res. 1970;4:141-147.
41. Daugherty A, Frayn KN, Redfern WS, Woodward B. The role of catecholamines in the production of ischaemia-induced ventricular arrhythmias in the rat in vivo and in vitro. Br J Pharmacol. 1986;87:265-277. [Medline] [Order article via Infotrieve]
42.
Toullec D, Pianetti P, Coste H, Bellevergue P,
Grand-Perret T, Ajakane M, Baudet V, Boissin P, Boursier E, Loriolle
F. The bisindolylmaleimide GF 109203X is a potent and selective
inhibitor of protein kinase C. J
Biol Chem. 1991;266:15771-15781.
43.
Bugge E, Ytrehus K. Ischaemic preconditioning is
protein kinase C dependent but not through stimulation of
-adrenergic or adenosine receptors in the isolated rat
heart. Cardiovasc Res. 1995;29:401-406. [Medline]
[Order article via Infotrieve]
44.
Mitchell MB, Meng X, Ao L, Brown JM, Harken AH,
Banerjee A. Preconditioning of isolated rat heart is mediated by
protein kinase C. Circ Res. 1995;76:73-81.
This article has been cited by other articles:
 |
|
 |
|
  T. Miura, S. Kawamura, H. Tatsuno, Y. Ikeda, S. Mikami, H. Iwamoto, T. Okamura, M. Iwatate, M. Kimura, Y. Dairaku, et al. Ischemic Preconditioning Attenuates Cardiac Sympathetic Nerve Injury via ATP-Sensitive Potassium Channels During Myocardial Ischemia Circulation, August 28, 2001; 104(9): 1053 - 1058. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Costa, N. J. Christensen, G. Farley, and I. Biaggioni NO modulates norepinephrine release in human skeletal muscle: implications for neural preconditioning Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2001; 280(5): R1494 - R1498. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Lochner, S. Genade, E. Tromp, T. Podzuweit, and J. A. Moolman Ischemic Preconditioning and the {beta}-Adrenergic Signal Transduction Pathway Circulation, August 31, 1999; 100(9): 958 - 966. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Takasaki, N. Adachi, K. Dote, S. Tsubota, T. Yorozuya, and T. Arai Ischemic preconditioning suppresses the noradrenaline turnover in the rat heart Cardiovasc Res, August 1, 1998; 39(2): 373 - 380. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||