(Circulation. 2001;103:1638.)
© 2001 American Heart Association, Inc.
Clinical Investigation and Reports |
Acetylcholine Release in Human Heart Atrium
Influence of Muscarinic Autoreceptors, Diabetes, and Age
From Medizinische Universitätsklinik, Innere Medizin IV (V.O., E.S., T.R., L.C.R.), and Herzchirurgie (F.B.), Freiburg, Germany.
Correspondence to Lars Christian Rump, MD, Innere Medizin IV, Hugstetter Str 55, D-79106 Freiburg, Germany. E-mail lcrump{at}med1.ukl.uni-freiburg.de
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Abstract |
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Background—An imbalance of sympathetic and parasympathetic drive to the heart is an important risk factor for cardiac death in patients with coronary heart disease, diabetes, and renal insufficiency. The amount of neurotransmitter released from peripheral autonomic nerves is modulated by presynaptic receptor systems. In analogy to
-autoreceptors on
sympathetic nerves, muscarinic autoreceptors activated by endogenous
acetylcholine may exist on parasympathetic nerves in the human
heart. Methods and Results—We developed a technique to study acetylcholine release from human atria and investigated muscarinic autoreceptor function. A pharmacological and molecular approach was used to characterize the subtype involved. Of the 5 muscarinic receptor subtypes cloned, only mRNA encoding for M2- and M3-receptors were detected. Potencies of several muscarinic antagonists against the release-inhibiting effect of the nonselective muscarinic agonist carbachol at the cardiac autoreceptor were correlated with published data for human cloned M1- through M5-receptors.
Conclusions—This analysis clearly indicates that acetylcholine release in human atria is controlled by muscarinic M2-receptors. Blockade of these receptors by atropine doubles the amount of acetylcholine released at a stimulation frequency of 5 Hz. In atria of patients >70 years of age and patients with late diabetic complications, acetylcholine release is reduced. Locally impaired cardiac acetylcholine release may therefore represent a pathophysiological link to sudden cardiac death in elderly and diabetic patients.
Key Words: coronary disease • nervous system, sympathetic • acetylcholine • receptors • diabetes mellitus
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Sympathetic overactivity plays a dominant prognostic role in myocardial infarction and heart failure of various causes, and the role of enhanced cardiac norepinephrine release is well described. Surprisingly, little is known about the role of the cholinergic nervous system, which is considered to be a functional counterpart of sympathetic influences to the heart.1 Parasympathetic activity is suppressed in heart failure,2 diabetes,3 and old age,4 and an imbalance of sympathetic and parasympathetic drive may contribute to the high incidence of sudden cardiac death in these patients. This view is mainly based on indirect measures of cardiac parasympathetic dysfunction such as altered heart rate variability5 and baroreflex sensitivity6 ; however, nothing precise is known about presynaptic mechanisms controlling cardiac acetylcholine release in humans. Parasympathetic nerve endings may have presynaptic receptors, which when activated inhibit acetylcholine release.7 8 9 The most important presynaptic receptor systems are those activated by endogenous ligands. In analogy to presynaptic
-autoreceptors on
sympathetic nerve
terminals,10 muscarinic
autoreceptors activated by neuronally released acetylcholine may exist
on parasympathetic nerves in the human heart. The existence of
presynaptic muscarinic autoreceptors may explain the clinical
observation of why low doses of the M1-selective
antagonist pirenzipine decrease heart rate in
humans.11 Five human
muscarinic receptors (M1 through
M5) have been
cloned,12 and one could
hypothesize that presynaptic and postsynaptic receptor subtypes differ.
Therefore, we determined the muscarinic autoreceptor subtype of
parasympathetic nerves regulating acetylcholine release in the human
heart and investigated whether diabetes or old age modifies
acetylcholine release. |
Methods |
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Patients and Tissue Sources
The study was approved by the Ethics Committee of the University of Freiburg. Atrial tissues were taken from patients undergoing open heart surgery for coronary bypass grafting (78 patients) or aortic valve replacement (18 patients). Specimens of
100 to 200 mg were harvested from the tip of the right auricle
during venous cannulation for extracorporal circulation. The age of the
patients (21 women; 75 men) averaged 67±1 years (range, 41 to 82).
Patients with diabetes were separated into groups according to the
presence (10 patients) or absence (8 patients) of diabetic
complications (nephropathy, polyneuropathy, and retinopathy). Diabetic
nephropathy was assumed in diabetic patients with a serum creatinine of
1.2 mg/dL and pathological microalbuminuria (>30 mg/24 h) or overt
proteinuria (>300 mg/24 h). Diagnosis of retinopathy and
polyneuropathy was based on diagnosed typical findings of funduscopy
and neurological examination, respectively.
Release Experiments
Connective tissue was removed and multiple segments
were prepared. Segments were incubated with
[3H]-choline (25 µCi, 0.31 nmol) between
platinum electrodes in Krebs-Henseleit solution gassed with 95%
O2 /5% CO2. During
incubation (75 minutes), field stimulation (40 V, 20 mA, 2 ms) was
applied every 5 seconds to increase uptake of
radioactivity.13 The
segments were then placed into superfusion chambers (volume, 250 µL).
Eight segments were superfused (2 mL/min) in parallel at 37°C.
Hemicholine (3 µmol/L) was added after 65 minutes to prevent reuptake
of [3H]-choline. After washout, twenty
3-minute fractions of the superfusion solution were collected. Two
stimulations (40 mA, 5 Hz, 2 minutes) were applied (S1 at 7 minutes, S2
at 45 minutes after start of collections). S1 served as reference
stimulation. The effect of drugs was tested by adding them 15 minutes
before S2. In those experiments in which a drug was present in both
stimulation periods (throughout), it was added 15 minutes before
S1.
Estimation of Radioactivity
The radioactivity of the 3-minute samples was
detected by liquid scintillation counting. Total tissue radioactivity
was determined at the end of each experiment after the tissue was
dissolved in Soluene (Packard).
Calculation of Results
The spontaneous outflow of radioactivity was
determined as the mean of radioactivity of 2 samples, collected before
and after electrical stimulation. The stimulation-induced (S-I) outflow
of radioactivity was calculated by subtracting the spontaneous outflow
of radioactivity from the radioactivity present in the three 3-minute
samples collected immediately after onset of stimulation
(Figure 1
). S-I outflow of radioactivity is subsequently
expressed as a percentage of the total tissue content of radioactivity
at the time of stimulation (fractional S-I outflow in S1 and S2; FR1,
FR2). When effects of drugs were tested, results were calculated as FR2
to FR1 ratios and expressed as percentages of the corresponding
controls. Antagonist potencies (pKB values) were
estimated according to Equation 4 of Furchgott.14
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Statistics
All data are expressed as mean±SEM. Data points were
always determined in duplicate; n gives the number of atria used in
each group. Data were analyzed by unpaired Student’s
t test or ANOVA where
appropriate. Values of P<0.05
were considered statistically significant.
RNA Extraction and Reverse
Transcription–Polymerase Chain Reaction
Atrial tissue was homogenized and total RNA was
isolated from the supernatant with the RNeasy Kit (Quiagen). DNA was
isolated by the same method with RNAase instead of DNAse. cDNA
synthesis and reverse transcription–polymerase chain reaction (RT-PCR)
were performed at 57°C and 35 cycles with the SuperScript
preamplification system (GIBCO BRL). Homologous primers for human
M1- through M5-receptors
were used
(Table 1). RT-PCR products were analyzed by
electrophoretic separation. For negative control, RT-PCR performed
without reverse transcriptase was used. Primer efficiency with genomic
DNA was performed under the same conditions. PCR products were cloned
in the pCR2.1-TOPO plasmide with the TOPO-TA cloning kit (Invitrogen)
and sequenced at the Human Genetic Institute (Freiburg, Germany).
Sequences were verified by BLAST search at the National Center for
Biotechnology Information.
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Drugs and Vehicles
Krebs-Henseleit solution (mmol/L) contained NaCl 118,
KCl 4.7, CaCl2 2.5, MgCl2
0.45, NaHCO3 25,
KH2PO4 1.03,
D-(+)-glucose 11.1,
disodium-EDTA 0.067, and ascorbic acid 0.07. The following drugs were
used: [3H]-choline (Amersham); atropine,
pirenzepine, himbacine, TTX, EGTA, and carbachol (all Sigma);
tropicamide and AFDX-116 (Tocris); and hemicholinum (ICI Chemicals).
Drugs were dissolved with either distilled water, DMSO, or
ethanol.
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Results |
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[3H]-Acetylcholine Release From Human Atria
Human atrial segments incubated with [3H-choline] accumulated 33 140±1170 dpm per milligram of tissue wet weight. Electrical field stimulation with 600 pulses at 5 Hz (Figure 1
) induced an increase of the fractional outflow of
radioactivity of 0.27±0.02% (n=49). Fractional outflow in the two
stimulation periods (S1, S2) was stable
(Figure 1), with a ratio (FR2/FR1) of 89±2.1% (n=49).
Tetrodotoxin (1 µmol/L) and omission of
Ca2+ abolished the release of S-I
radioactivity
(Figure 1). Thus, the S-I release of radioactivity from human
atrial tissue could be taken as an index of
[3H]-acetylcholine release, as shown
previously for other
species.9
Modulation of Acetylcholine Release by
Muscarinic Autoreceptors in Human Atria
The nonselective muscarinic agonist carbachol (0.1 to
10 µmol/L) inhibited the release of radioactivity concentration
dependently (EC50 of 0.9 µmol/L) and at a
maximum of 78%
(Figure 2). Atropine (0.1 µmol/L), a nonselective
muscarinic antagonist, when present throughout superfusion, shifted the
dose-response curve of carbachol to the right
(Figure 2). Atropine by itself enhanced the release of
radioactivity dose dependently. To further characterize the receptor
subtype involved, dissociation constants (pKB
values) of 5 muscarinic antagonists were determined
(Table 2) against carbachol and plotted against affinity
estimates of cloned human muscarinic receptors. Whenever possible,
pKB
values15 16 were
taken for correlation. In the case that pKB
values were not available, pKi values were used
instead.17 18
There was a significant correlation for the M2-
and M4-receptors, with the best correlation
coefficient calculated for the M2-receptor
(Figure 3). This view is supported by the rank order of
potencies:
atropine>himbacine>AFDX-116tropicamide>pirenzepine,
compatible with the involvement of an M2
receptor.
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RT-PCR of Muscarinic Receptors in Human
Atria
Under the conditions applied, only significant mRNA
levels of M2- and
M3-receptor subtypes were found
(Figure 4). The M2-receptor band was
more prominent than the M3-receptor band. RT-PCR
without reverse transcriptase (-RT;
Figure 4) showed that the RT-PCR products obtained
originated from mRNA. PCR of genomic DNA revealed bands for 5
muscarinic receptors (data not shown), proving the specificity of the
primers.
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Influence of Age and Late Diabetic
Complications on Acetylcholine Release From Human Atria
Uptake of radioactivity after incubation with
[3H]-choline was similar in all age groups
(Figure 5). However, S-I release of
[3H]-acetylcholine decreased with age.
Segments of patients >70 years of age showed a lower release of
[3H]-acetylcholine than atria of patients
70 years of age. Uptake of radioactivity after incubation with
[3H]-choline was similar in patients with
or without diabetes but lower in diabetic patients with late
complications
(Figure 6). Atrial segments of patients with late diabetic
complications also had a significant lower fractional release of
[3H]-acetylcholine than control subjects
and diabetic patients without late complications
(Figure 6). The average age of the 3 groups was not different
(63±3, 68±2, and 66±1 years).
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Discussion |
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A suppressed parasympathetic nervous system of the heart may play an important role for prognosis in diseases such as heart failure, myocardial infarction, and diabetes. In this study, we established a method to investigate acetylcholine release and its modulation by inhibitory muscarinic autoreceptors from human cardiac tissue. Because there was indirect evidence to suggest an impaired release of acetylcholine release in old age11 and diabetes,19 we took advantage of our technique to look into this matter.
Acetylcholine Release From Human Atria
Labeling of the cholinergic transmitter stores
with [3H]-choline taken up by cholinergic
nerves and synthesized to
[3H]-acetylcholine by choline
acetyltransferase has been used in atrial tissue of guinea pigs, rats,
and
mice20 21 22
but not in atrial tissue of humans. We modified this technique for
human atria and showed a significant outflow of
[3H] activity in response to electrical
field stimulation. Within 1 hour after obtaining the tissue in the
operation theater, experiments were started, which then lasted for 3
hours. This short time interval makes it unlikely that effects of
denervation may have altered uptake and release functions of
parasympathetic nerve endings. The S-I release of radioactivity was
completely tetrodotoxin sensitive and dependent on extracellular
Ca2+, fulfilling the criteria of exocytotic
release processes. Therefore, the S-I outflow of radioactivity from
human atria preincubated with [3H]-choline
was taken as an index of endogenous acetylcholine
release.
Characterization of Cardiac Inhibitory
Autoreceptor
The amount of acetylcholine released at parasympathetic
neuroeffector junctions in the heart is primarily regulated by the
central nervous system integrating afferent inputs from the
baroreceptor reflex and cardiopulmonary mechanoreceptors and
chemoreceptors. However, postganglionic cholinergic nerve endings have
presynaptic receptors, which, when activated by endogenous
acetylcholine, inhibit the release of cardiac acetylcholine. In several
animal species, such muscarinic autoreceptors have been demonstrated in
cardiac tissue. These receptors involved in autoinhibition of
acetylcholine release are of the M1-subtype in
chicken heart7 and of the
M2-subtype in guinea pig, rat, and rabbit
heart.7 8 9
Functional studies suggest the existence of presynaptic
M1-receptors in humans. It was postulated that
the decrease of heart rate by low concentrations of the preferential
M1-receptor antagonist pirencepine is due to
blockade of presynaptic M1-receptors enhancing
acetylcholine release.11
However, binding studies revealed only the existence of
M2-receptors in the human heart.23 To clarify this
situation, we used two different approaches: pharmacological
characterization with Equation 4 of Furchgott14 and molecular
characterization with RT-PCR. First, potencies
(pKB values) of 5 muscarinic receptor
antagonists against the release-inhibiting effect of the nonselective
agonist carbachol were determined and compared with
pKB/pKi values of cloned human muscarinic
receptors.15 16 17 18
The highest correlation coefficient of 0.98 was determined for the
M2-subtype, and the slope of the regression was
close to unity. pKB values obtained for human
autoreceptors were 10-fold lower than those for the cloned receptors.
This difference is expected because in our study, the antagonists used
had to compete with carbachol and endogenous acetylcholine for the
presynaptic autoreceptor. The role of endogenous acetylcholine
activating presynaptic autoreceptors is clearly demonstrated by the
marked enhancement of acetylcholine release induced by atropine. Taken
together, the pharmacological characterization suggests the involvement
of presynaptic autoreceptor of the M2- but not
the M1-subtype in human atria. This is in
accordance with a previous study in human
trachea.24 In contrast, at
parasympathetic ganglia, the
M1-subtype,25
in urinary bladder the
M3-subtype26
and in detrusor the M4-subtype, has been
described to modulate acetylcholine
release.27
Pharmacological characterization may lead to a false interpretation because of imperfect specificity of muscarinic receptor antagonists available. However, the operation of cardiac muscarinic M2-autoreceptors is supported by results with RT-PCR. With the use of homologous primers, RT-PCR amplified only cDNA encoding for M2- and M3-receptor subtypes. The detection of RT-PCR products for M2-receptors fits well with our pharmacological characterization. However, it is likely that the RT-PCR signal for M2-receptors comes to some extent from mRNA encoding for postsynaptic M2-receptors. M3-receptors have as yet not been found in human heart, but there were some indications from in vitro and in vivo studies that an additional muscarinic receptor, different from the M2-subtype, may exist in human heart.11 M3-receptors have already been demonstrated in chicken28 and dog heart.29 However, our pharmacological analysis excludes presynaptic M3-receptors in human atria. M1-, M4-, and M5-receptors were excluded by RT-PCR. With the pharmacological and molecular evidence taken together, it is feasible that in the human heart, acetylcholine release is modulated exclusively by M2-autoreceptors.
Influence of Age and Late Diabetic
Complications
The physiological process of aging as well as advanced
stages of diabetes have been associated with profound changes in
autonomic function. In older age, circulating plasma norepinephrine
levels are
increased.30 31
More recently, an imbalance between sympathetic and parasympathetic
influences to the heart in diabetes and old age have been suggested on
the basis of heart rate variability
studies.5 This dysregulation
may be due to defective afferent and efferent signaling processes;
however, in addition, alterations of local presynaptic release
mechanisms of acetylcholine may be involved. It has already been shown
that muscarinic receptor density and function are decreased in old
age.11 The concept of a
locally altered parasympathetic nervous function is now supported by
the present in vitro study, which demonstrates that cardiac
acetylcholine release from parasympathetic nerves is decreased in
diabetes and old age. As for diabetes, this impairment was observed
only in patients with advanced stages of the disease (nephropathy,
retinopathy, neuropathy). This fits well with a study in diabetic
impotent men, which showed that synthesis and release of acetylcholine
in corporeal tissue was reduced and worsened with the duration of
diabetes.32 Furthermore,
animal studies investigating acetylcholine release from phrenic nerve
terminals33 and brain
slices34 in
streptozotocin-induced diabetes in rats support the concept of locally
altered parasympathetic regulation in diabetes. In the present study,
the uptake of [3H]-choline was reduced in
atria of diabetic patients with late complications. This may reflect a
reduced density of parasympathetic innervation or reduced ability of
[3H]-acetylcholine generation. However,
fractional release calculated as a percentage of the total accumulated
tissue radioactivity was also diminished, suggesting an additional
functional component. This appears to be true also for atria of
patients >70 years of age. Uptake of
[3H]-choline was similar to atria of
younger patients; however, S-I release of acetylcholine was reduced.
One possible explanation is that presynaptic
M2-autoreceptor function is upregulated, leading
to reduced acetylcholine release, as shown for rat diabetic
lungs.35 Taken together,
this is the first evidence to show impaired cardiac acetylcholine
release in diabetics and elderly people. This impaired local
parasympathetic activity may be an important risk factor for sudden
cardiac death in these groups of patients. Furthermore, alterations of
presynaptic modulation of acetylcholine release must be taken into
account when interpreting results of heart rate variability studies, in
which pharmacological manipulations are used to assess vagal
activity.
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Acknowledgments |
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This study was supported by the Deutsche Forschungsgemeinschaft (Schw 784/1–2) and Zentrum Klinische Forschung 2, University Hospital Freiburg. We thank Petra Stunz for excellent technical assistance.
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Footnotes |
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Drs Oberhauser and Schwertfeger contributed equally to this work.
Received September 27, 2000; revision received December 12, 2000; accepted December 13, 2000.
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G. Takagi, K. Asai, S. F. Vatner, R. K. Kudej, F. Rossi, A. Peppas, I. Takagi, R. R. G. Resuello, F. Natividad, Y.-T. Shen, et al. Gender differences on the effects of aging on cardiac and peripheral adrenergic stimulation in old conscious monkeys Am J Physiol Heart Circ Physiol, July 11, 2003; 285(2): H527 - H534. [Abstract] [Full Text] [PDF]
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J. Szilvassy, I. Sziklai, P. Horvath, M. Szilasi, J. Nemeth, P. Kovacs, and Z. Szilvassy Feeble bronchomotor responses in diabetic rats in association with decreased sensory neuropeptide release Am J Physiol Lung Cell Mol Physiol, May 1, 2002; 282(5): L1023 - L1030. [Abstract] [Full Text] [PDF]
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