Department of Pharmacology, George Washington University, Washington, DC 20037
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ABSTRACT |
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Wang, Jijiang, Mustapha Irnaten, and David Mendelowitz. Agatoxin-IVA-Sensitive Calcium Channels Mediate the Presynaptic and Postsynaptic Nicotinic Activation of Cardiac Vagal Neurons. J. Neurophysiol. 85: 164-168, 2001. Whole cell currents and miniature glutamatergic synaptic events (minis) were recorded in vitro from cardiac vagal neurons in the nucleus ambiguus using the patch-clamp technique. We examined whether voltage-dependent calcium channels were involved in the nicotinic excitation of cardiac vagal neurons. Nicotine evoked an inward current, increase in mini amplitude, and increase in mini frequency in cardiac vagal neurons. These responses were inhibited by the nonselective voltage-dependent calcium channel blocker Cd (100 µM). The P-type voltage-dependent calcium channel blocker agatoxin IVA (100 nM) abolished the nicotine-evoked responses. Nimodipine (2 µM), an antagonist of L-type calcium channels, inhibited the increase in mini amplitude and frequency but did not block the ligand gated inward current. The N- and Q-type voltage-dependent calcium channel antagonists conotoxin GVIA (1 µM) and conotoxin MVIIC (5 µM) had no effect. We conclude that the presynaptic and postsynaptic facilitation of glutamatergic neurotransmission to cardiac vagal neurons by nicotine involves activation of agatoxin-IVA-sensitive and possibly L-type voltage-dependent calcium channels. The postsynaptic inward current elicited by nicotine is dependent on activation of agatoxin-IVA-sensitive voltage-dependent calcium channels.
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INTRODUCTION |
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The control of heart
rate and cardiac function is determined primarily by cardiac vagal
neurons that are located in the nucleus ambiguus (NA) and the dorsal
motor nucleus (DMNX) of the vagus (Izzo et al. 1993;
Standish et al. 1994
, 1995
; Taylor et al.
1999
). These neurons dominate the neural control of heart rate
under normal conditions and also determine the prognosis of many
pathological cardiac challenges (Vanili and Schwartz
1990
). In each respiratory cycle, the heart beats more rapidly
in inspiration and slows during postinspiration and expiration
(referred to as respiratory sinus arrhythmia). Cardiorespiratory
interactions do not seem to occur between sensory neurons at their
first central synapses in the nucleus tractus solitarius (NTS),
suggesting cardiorespiratory interactions occur later in the reflex
pathways perhaps within the nucleus ambiguus (Mifflin et al.
1988
). With the discovery of diverse types of nicotinic
acetylcholine receptors (nAChRs) in the CNS, particularly the evidence
that nAChRs exist in synaptic terminals in NA and DMNX
(Winzer-Serhan and Leslie 1997
), acetylcholine as a
neurotransmitter is now suggested to be responsible for the respiratory
modulation of heart rate and may be involved in many cardiorespiratory
diseases, including sudden infant death (Feldman and Buccafusco
1993
; Florez et al. 1990
; Loewy and Spyer
1990
; Mallard et al. 1999
; Panigrahy et
al. 1997
; Slotkin 1998
).
Previous studies from this laboratory have described the activation of
cardiac preganglionic vagal neurons by nicotine and acetylcholine
(Mendelowitz 1998; Neff et al. 1995
,
1998a
). This activation involves presynaptic nAChRs that
increase the frequency of spontaneous glutamatergic miniature synaptic
events (minis) and postsynaptic nAChRs that enhance
non-N-methyl-D-aspartate (NMDA) currents and
evoke a direct ligand gated inward current. The increase of mini
frequency was blocked by
-bungarotoxin (
-Bgtx), a selective
antagonist of nAChRs that contain the
-7 gene product (Clarke
1992
; Couturier et al. 1990
), suggesting the
existence of these types of nAChRs in the presynaptic terminals of
neurons that project to cardiac vagal neurons.
The mechanisms by which -Bgtx sensitive nAChRs increase the
frequency of spontaneous release of transmitter are unclear.
-Bgtx-sensitive nAChRs have a high permeability to calcium, are preferentially localized, and are clustered at presynaptic sites (Amador and Dani 1995
; Castro and Albuquerque
1995
; Clarke 1992
; Couturier et al.
1990
; Zhang et al. 1996
). One possibility is that the calcium influx elicited from activation of the nAChR channel
is sufficient to cause spontaneous transmitter release. Another
possibility is that the spontaneous transmitter release depends on
nAChR-evoked presynaptic depolarization and subsequent activation of
presynaptic voltage-dependent calcium channels (VDCCs). This second
mechanism has been shown to be involved in the enhancement of
spontaneous GABA release from neuron terminals within chick lateral
spiriform and ventral lateral geniculate (Tredway et al. 1999
). It is also not known if VDCC are involved in the
postsynaptic enhancement of non-NMDA currents or the direct
ligand-gated inward current elicited by nicotine in cardiac vagal neurons.
The present study investigates whether VDCCs play a role in the presynaptic and postsynaptic nicotinic activation of cardiac preganglionic vagal neurons and the identity of the specific VDCC subtype(s) involved. This study indicates that nicotine-evoked increase in presynaptic transmitter release does involve presynaptic VDCCs, and among the subtypes, agatoxin-IVA-sensitive channels predominate with L type channels playing a minor role. Unexpectedly, the same VDCC subtypes are responsible for the postsynaptic enhancement of non-NMDA currents on activation of nAChRs.
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METHODS |
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Terminals of preganglionic vagal cardiac neurons are located
mostly in the fat pads at the base of the heart in rats
(Standish et al. 1994). In an initial surgery, rats of
6- to 12-days old were anesthetized with methoxyflurane and hypothermia
and received a right thoracotomy. The heart was exposed, and rhodamine
(XRITC, Molecular Probes) was injected into the pericardial sac. On the day of experiment (3-7 days later), the animals were anesthetized with
methoxyflurane and killed by rapid cervical dislocation. All animal
procedures were performed in compliance with the institutional guidelines at George Washington University and are in accordance with
the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association and the National Institutes of Health
publication "Guide for the Care and Use of Laboratory Animals." The
hindbrain was removed and placed for 1 min in cold (0-2°C) buffer of
the following composition (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 5 glucose, and 10 HEPES and was
continually gassed with 100% O2. The medulla was
then cut in sections of 250-µm thickness using a vibratome. Slices
were mounted in a perfusion chamber and submerged in the perfusate of
following composition (in mM): 125 NaCl, 3 KCl, 2 CaCl2, 26 NaHCO3, 5 dextrose, and 5 HEPES, constantly bubbled with gas (95%
O2-5% CO2), and maintained
at pH 7.4. Calcium channel antagonists were directly perfused into the
chamber at the following concentrations. CdCl2
(100 µM) was dissolved directly into the perfusate; nimodipine was
dissolved in ethanol for a stock solution of 2 mM, and diluted to a
final concentration of 2 µM. Conotoxin GVIA, agatoxin IVA, and
conotoxin MVIIC were prepared immediately before use and their
concentrations were 1 µM, 100 nM, and 5 µM, respectively.
Individual vagal cardiac neurons were identified by the presence of the
fluorescent tracer. These identified vagal cardiac neurons were then
imaged with differential interference contrast (DIC) optics, infrared
illumination, and infrared-sensitive video-detection cameras to gain
better spatial resolution and to visually guide and position the patch
pipette onto the surface of the identified neuron. The pipette was
advanced until obtaining a seal over 1 G between the pipette tip and
the cell membrane of the identified neuron. The membrane under the
pipette tip was then ruptured with a brief suction to obtain whole cell
patch-clamp configuration, and the cell was voltage-clamped at a
holding potential of
80 mV. Picrotoxin (100 µM), strychnine (1 µM), prazosine (10 µM), D-2-amino-5-phosphonovalerate
(50 µM), and tetrodotoxin (TTX, 1 µM) were infused to prevent
GABAergic, glycinergic,
1-adrenergic, glutaminergic NMDA postsynaptic currents and to prevent polysynaptic pathways, respectively. The pipettes were filled with a solution consisting of (in MM) 130 K+ gluconate, 10 HEPES,
10 EGTA, 1 CaCl2, and 1 MgCl2.
Nicotine (1 µM) was pressure injected directly onto the neuron by a
micropipette positioned directly above the neuron for 20-40 s. After
application, a brief (approximately 10 s) negative pressure pulse
was applied to limit any diffusion of nicotine out of the micropipette.
The slice was then perfused with a calcium channel antagonist for 20 min. At the end of this 20-min period, nicotine was reapplied in the
continued presence of the antagonist. A 20-min delay was used to
minimize any desensitization of the cell by nicotine. Analysis of
spontaneous postsynaptic events was performed using MiniAnalysis
(Synaptosoft, version 4.3.1) with an amplitude threshold of 6 pA.
Different groups of neurons were examined for each toxin. Responses to
nicotine during simultaneous application of the toxin were
statistically compared with the responses from control nicotine
applications using student's paired t-test. The data are
presented as means ± SE, with one asterisk signifying differences
of P 0.05, and two asterisks for P
0.01.
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RESULTS |
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Nicotine evoked an inward current in cardiac vagal neurons as well as dramatic increases in both the frequency and the amplitude of the minis (Fig. 1, left). CdCl2 inhibited, but did not completely abolish, this nicotinic activation of cardiac vagal neurons (Fig. 1, right). In the presence of CdCl2, nicotine did not induce any significant change in baseline current. Nicotine significantly increased mini frequency and amplitude when accompanied with CdCl2, but these responses were significantly reduced by 75.5 and 83.9%, respectively, compared with control responses.
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Since the nonspecific VDCC blocker CdCl2
inhibited the pre- and postsynaptic nicotine-evoked responses, we next
examined which VDCCs were responsible for these nicotine-mediated
changes. The specific P-type VDCC blocker -agatoxin IVA (100 nM)
completely abolished all presynaptic and postsynaptic nicotinic
activation of cardiac vagal neurons.
-Agatoxin IVA (Fig.
2, right) blocked the
nicotine-induced inward current, increases in mini frequency and mini
amplitude by 98.2, 87.0, and 99.6%, respectively, compared with
control responses (Fig. 2, left).
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In contrast the specific L-type VDCC blocker nimodipine inhibited the nicotine-evoked increases in mini amplitude and frequency but did not significantly inhibit the nicotine-eicited ligand-gated inward current (Fig. 3). The nicotine-evoked increases in mini frequency and amplitude were not statistically significant from baseline values when accompanied by nimodipine. However, the nicotine-evoked inward current during nimodipine remained statistically significant and was only attenuated by 46.6% from the control nicotine response.
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-Conotoxin MVIIC and
-conotoxin GVIA did not alter the nicotinic
activation of cardiac vagal neurons. In the presence of
-conotoxin
MVIIC (5 µM), the nicotine-induced increases of the frequency and the
amplitude of the minis, as well as the inward current, were not
significantly different from control responses. Similarly
-conotoxin
GVIA (1 µM) did not alter the pre- and postsynaptic nicotine-evoked
responses. The effects of each calcium channel antagonist on the
nicotinic activation of cardiac vagal neurons are summarized in Fig.
4.
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DISCUSSION |
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Recent studies of cardiac vagal neurons in the nucleus ambiguus
(NA) have demonstrated that these neurons are intrinsically silent and
do not exhibit spontaneous pacemaker like activity (Mendelowitz
1996). In agreement with these in vitro results, the relatively
few in vivo studies that have successfully examined preganglionic
cardiac neurons with extracellular electrodes have also found that most
of these neurons (identified by antidromic stimulation) were silent
(Giby et al. 1984
). These results suggest that the tonic
vagal activity that is normally present in unanesthesized animals
depends on excitatory synaptic contacts to initiate and maintain vagal
cardiac activity.
Two important transmitters that excite cardiac vagal neurons are
glutamate and acetylcholine. One of the more functionally important
pathways for blood pressure regulation is the glutamatergic pathway
from the NTS (Neff et al. 1998b; Taylor et al.
1999
; Willis et al. 1996
). The NTS is the site
of the first central synapse for visceral sensory neurons, including
arterial baroreceptors, and the monosynaptic pathway from the NTS to
cardiac vagal neurons most likely plays an essential role in
cardiovascular reflex control. Acetylcholine is also an important
transmitter innervating cardiac vagal neurons, and this input likely
play an important role in cardiorespiratory interactions (Giby
et al. 1984
). Our previous work has shown that nicotine, but
not muscarinic agonists, activates postsynaptic receptors and a
depolarizing inward current in vagal cardiac neurons (Neff et
al. 1998a
). In addition, nicotine acts at different pre- and
postsynaptic sites to facilitate glutamatergic neurotransmission
(Mendelowitz 1998
; Neff et al. 1998a
).
Presynaptic nicotinic receptors increase the frequency of transmitter
release, and are sensitive to block by
-bungarotoxin. Nicotine also
elicits an augmentation of postsynaptic non-NMDA currents. These
nicotinic responses may serve to directly depolarize cardiac neurons as well as augment the glutamatergic input to cardiac vagal neurons both
pre- and postsynaptically during the postinspiratory phase of the
respiratory cycle. These latter two effects may constitute mechanisms
by which cholinergic respiratory neurons gate or facilitate the
baroreflex during postinspiration.
This work demonstrates that VDCCs play a critical role in mediating the
nicotinic facilitation of glutamatergic responses both pre- and
postsynaptically. The nonspecific VDCC blocker Cd and the specific VDCC
blocker agatoxin IVA inhibited the nicotine-evoked increase in mini
frequency. This suggests that although the presynaptic 7 subunit
containing nicotinic receptors is responsible for the increase in
transmitter discharge and is highly permeable to calcium, the increase
in mini frequency is dependent on concurrent activation of VDCCs, and
in particular agatoxin-IVA-sensitive calcium channels. It therefore
seems likely that activation of
7 subunit containing presynaptic
nicotinic receptors is sufficient to evoke depolarization at the
presynaptic terminals to activate VDCCs but cannot independently increase presynaptic calcium to sufficient levels to evoke transmitter release.
The postsynaptic facilitation of non-NMDA currents and the direct
ligand-gated inward current elicited by nicotine are also dependent on
activation of VDCCs. The subtype of nicotinic receptors located
postsynaptically in cardiac vagal neurons that are responsible for
these postsynaptic responses are unknown. The postsynaptic nicotinic
receptors in cardiac vagal neurons are insensitive to -bungarotoxin
and therefore do not contain the
7 subunit (Neff et al.
1998a
). Surprisingly however, the postsynaptic nicotinic responses are also dependent on VDCCs and can be blocked by Cd as well
as agatoxin IVA. This suggests that both the nicotinic facilitation of
non-NMDA currents, as well as the direct postsynaptic inward currents,
require activation of VDCCs and in particular the agatoxin
IVA-sensitive VDCC. It is possible that these nicotinic responses rely
on a Ca-dependent or other second messenger that is only activated when
postsynaptic VDCCs are at least partially open.
Nimodipine partially inhibited the nicotine-evoked increase in mini
frequency and amplitude but did not significantly alter the direct
inward current. One possibility is that L-type VDCCs are involved in
enhancing glutamatergic transmission; but another possibility is that
nimodipine directly inhibits these nicotinic receptors. Other studies
have provided evidence that L-type VDCC antagonists may directly alter
nicotinic receptors (Donnelly-Roberts et al. 1995).
Since the subunit composition and the full pharmacological profile of
the nicotinic receptors located postsynaptically in cardiac vagal
neurons are unknown, we cannot rule out the possibility that nimodipine
directly inhibits these nicotinic receptors. It is highly unlikely that
the inhibition of the nicotinic responses with Cd or agatoxin are due
to direct effects on the nicotinic receptors since the concentration of
Cd and agatoxin used in this study (100 µM and 100 nM, respectively)
have been shown to have no effect on nicotinic receptors
(Donnelly-Roberts et al. 1995
).
It is somewhat surprising that the nicotine-evoked responses are
blocked by agatoxin IVA but are unaltered by -conotoxin MVIIC. In
many neurons, studies have shown that agatoxin IVA and
-conotoxin
MVIIC block the same P/Q type VDCCs (for review, see Meir et al.
1999
). However, there are some neurons in which a population of
VDCCs are blocked by agatoxin IVA but are insensitive to
-conotoxin
MVIIC. For example, in crayfish nerve terminals (Wright et al.
1996
), peptidergic neurons (Garcia-Colunga et al. 1999
), and teleost retinal cone horizontal neurons
(Pfeiffer-Linn and Lasater 1996
), agatoxin IVA nearly
completely blocked VDCCs while these VDCCs were not altered by
-conotoxin MVIIC. In humans, there may be developmental changes in
the sensitivity of VDCCs to blockers since in a 10-mo-old child, but
not in adults, hippocampal granule cells are agatoxin IVA sensitive but
are insensitive to
-conotoxin MVIIC (Beck et al.
1997
). It has recently been suggested that there is a much
greater diversity of voltage-dependent calcium channel subtypes than
previously recognized and can be distinguished with the L, N, P, Q, and
R toxin-based classification (Burley and Dolphin 2000
).
In summary, the nicotine-evoked inward current and both the pre- and
postsynaptic enhancement of glutamatergic synapses in cardiac vagal
neurons is dependent of activation of agatoxin-IVA-sensitive VDCCs.
L-type, but not N- or -conotoxin MVIIC-type, VDCCs may also be
involved. It therefore seems likely that for nicotine to enhance
glutamatergic pathways to cardiac vagal neurons, the presynaptic
neurons need to depolarize sufficiently to activate VDCCs, and the
VDCCs are also involved in the postsynaptic facilitation.
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ACKNOWLEDGMENTS |
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The technical assistance of K. Peterson, C. Evans, and A. Elibero is gratefully acknowledged.
This work was supported by National Heart, Lung, and Blood Institute Grants HL-49965 and HL-59895.
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FOOTNOTES |
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Address for reprint requests: D. Mendelowitz, Dept. of Pharmacology, George Washington University, 2300 Eye St. N.W., Washington, DC 20037 (E-mail: dmendel{at}gwu.edu).
Received 7 July 2000; accepted in final form 2 October 2000.
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REFERENCES |
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