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Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan, Taiwan (C.C.H., K.S.H.); and Center for Neuroscience, National Sun Yat-sen University, Kaohsiung, Taiwan (S.H.H.C.)
Received March 10, 2003; accepted May 12, 2003
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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-conotoxin GVIA selectively blocked the spermine NONOate-induced
synaptic potentiation. These results suggest that NO acts presynaptically to
elicit a synaptic potentiation on the RVLM neurons through an enhancement of
presynaptic N-type Ca2+ channel activity leading to
facilitating glutamate release. The presynaptic action of NO is mediated by a
cGMP/PKG-coupled signaling pathway.
). Both
electrophysiological and anatomical studies showed that a subset of RVLM
neurons directly innervates preganglionic vasomotor neurons in the
intermediolateral cell columns of the spinal cord, which are involved in the
regulation of vasomotor and cardiac tone
(Morrison et al., 1991;
Deuchars et al., 1995). In
addition, the RVLM neurons are tonically active in vivo and receive convergent
excitatory and inhibitory inputs from multiple sources
(Mtui et al., 1995;
Guyenet et al., 1996).
However, little is known regarding the physiological regulation of the tonic
excitation of these neurons.
Nitric oxide (NO), an unstable diatomic radical synthesized from
L-arginine by nitric-oxide synthase (NOS), is recognized as an
important signaling molecule mediating a variety of physiological processes,
including synaptic transmission and synaptic plasticity
(Garthwaite, 1991). In
addition, considerable evidence suggests that NO is also involved in central
cardiovascular regulation and that one of the potential sites in the central
nervous system for NO to exert its modulation action on cardiovascular
functions is the RVLM (Hakim et al.,
1995; Zanzinger et al.,
1995; Hirooka et al.,
1996; Kagiyama et al.,
1997; Chan et al.,
2001). Studies using immunohistochemistry, NADPH-diaphorase
staining, or autoradiography further demonstrate the presence of neuronal NOS
(nNOS), inducible NOS (iNOS), and endothelial NOS in the RVLM
(Vincent and Kimura, 1992;
Chang et al., 2001). We
demonstrated recently (Chan et al.,
2001) that both nNOS and iNOS in the RVLM are tonically active
under physiological conditions at the levels of functional expression and
molecular synthesis. More importantly, we showed that the prevalence of nNOS
over iNOS activity at the RVLM and the associated dominance of
sympathoexcitation over sympathoinhibition underlie the maintenance of
sympathetic vasomotor outflow and stable arterial pressure by the endogenous
NO.
An important corollary to these findings is how NO exerts its modulatory
action on RVLM neurons to regulate the cardiovascular function. Because
glutamate is the major neurotransmitter in tonic excitatory drive of RVLM
neurons (Sved et al., 2001),
we have therefore examined the role of NO on the glutamatergic transmission of
RVLM neurons in brainstem slice preparations from immature rats using
whole-cell patch-clamp recordings. Our results suggest that NO acts
presynaptically to elicit a stable potentiation of synaptic transmission by
means of a mechanism involving the NO-sensitive sGC/cGMP/PKG-coupled signaling
cascade, and these effects may have resulted in a sympathoexcitatory effect of
NO in the RVLM.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). In
brief, rats were killed by decapitation under halothane anesthesia, and
coronal brainstem slices (200 µm thick) were prepared using a vibrating
microtome (Leica VT1000S; Leica, Nussloch, Germany). The slices were placed in
a storage chamber of artificial cerebrospinal fluid (ACSF) oxygenated with 95%
O2/5% CO2 and kept at room temperature for at least 1 h
before recording. The composition of the ACSF solution was 117 mM NaCl, 4.7 mM
KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 25 mM
NaHCO3, 1.2 mM NaH2PO4, and 11 mM glucose at
pH 7.3 to 7.4 and equilibrated with 95% O2/5% CO2.
Patch-Clamp Recordings. For patch-clamp recording, slices were
transferred to a recording chamber and fixed at the glass bottom of the
chamber with a nylon grid on a platinum frame. The chamber consisted of a
circular well of a low volume (1–2 ml) and was perfused constantly at
room temperature (24–26°C) at a speed of 2 to 3 ml/min. Visualized
whole-cell patch-clamp recordings of synaptically evoked EPSCs and spontaneous
EPSCs (sEPSCs) were conducted using standard methods
(Huang et al., 2001). The RVLM
was recognized in the slice as an area ventral to the nucleus ambiguous that
appears as a slightly dark area in a freshly prepared medullary slice and
lateral to the paragigantocellular nucleus at a level rostral to the area
postrema (Hwang and Dun,
1998). The RVLM neurons were visualized throughout the experiment
with an upright microscope (BX50WI; Olympus, Tokyo, Japan) equipped with a
water-immersion 40x objective using Nomarski-type differential
interference contrast optics combined with infrared videomicroscopy. Patch
pipettes were pulled from borosilicate capillary tubing and heat polished. The
electrode resistance was typically 4 to 5 M
. The composition of
intracellular solution was 115 mM K-gluconate, 20 mM KCl, 10 mM HEPES, 2 mM
MgCl2, 10 mM EGTA, 3 mM Na2ATP, 0.3 mM
Na3GTP, 5 mM QX-314, and sucrose to bring osmolarity to 290 to 295
mOsM and pH to 7.3. After a high resistance seal (> 2 G before
breaking into whole-cell mode) was obtained, suction was applied lightly
through the pipette to break through the membrane. The cell was then
maintained at -70 mV for several minutes to allow diffusion of the internal
solution into the cell body and dendrites. Recordings were made using an
Axopatch 200B (Axon Instruments, Union City, CA) amplifier. Electrical signals
were low-pass filtered at 2 kHz and digitized at 4 to 10 kHz using a Digidata
1200B interface. An Intel Pentium-based computer with pCLAMP software (version
8.0; Axon Instruments) was used for on-line acquisition and off-line analysis
of the data. For measurement of synaptically evoked EPSCs, a bipolar stainless
steel stimulating electrode was applied to a site 300 to 450 µm dorsal to
the recorded neurons; the superfusate routinely contained bicuculline
methiodide (10 µM) and strychnine hydrochloride (0.5 µM) to block
inhibitory synaptic responses. The strength of synaptic transmission was
quantified by measuring the amplitude of EPSCs over a 0.5- to 2-ms window
concentrated around the peak. Series resistance (Rs) was calculated according
to Rs = 10 mV/I, where I was the peak of transient current (filtered with 10
kHz) evoked by the 10-mV testing pulse when the pipette capacitance was
compensated fully. Only cells demonstrating <25 M series resistance
(usually 10–20 M) were used in these experiments.
sEPSCs comprise both action potential-dependent and -independent synaptic events observed in the absence of synaptic stimulation. In the present study, sEPSCs were recorded from RVLM neurons held in voltage clamp at a potential of -70 mV in the presence of bicuculline methiodide (10 µM) and strychnine hydrochloride (0.5 µM) and analyzed off-line using a commercially available software (Mini Analysis 4.3; Synaptosoft, Leonia, NJ). The software detects events based on amplitudes exceeding a threshold set just above the baseline noise of the recording. The threshold for detection was set at -3 pA. All detected events were re-examined and accepted or rejected based on subjective visual examination. The program then measured amplitudes and intervals between successive detected events. Frequencies were calculated by dividing the total number of detected events by the total time sampled. Periods of 5 to 10 min were analyzed for each pharmacological treatment, and these recordings were visually inspected to allow for the removal of artifacts. Cumulative probability plots were constructed to compare drug effects on the distribution of amplitude and interevent intervals from sEPSCs. Amplitude histograms were binned in 1-pA intervals.
Drug Application. All drugs were applied by dissolving them to the
desired final concentrations in the ACSF and by switching the perfusion from
control ACSF to drug-containing ACSF. Appropriate stock solutions of drugs
were made and diluted with ACSF just before application. 7-NI, nimodipine, and
ODQ were dissolved in dimethyl sulfoxide stock solutions and stored at
-20°C until the day of experiment. Other drugs used in this study were
dissolved in distilled water. The concentration of DMSO in the perfusion
medium was 0.1%, which alone had no effect on the basal synaptic transmission.
L-Arginine, 7-NI, ODQ, SIN-1, nimodipine, -conotoxin GVIA,
6-cyano-7-notroquinoxaline-2,3-dione (CNQX), spermine NONOate,
D-(-)-
-amino-5-phosphonopentanoic acid (D-APV),
and bicuculline methiodide were purchased from Tocris Cookson (Bristol, UK);
D-arginine, strychnine hydrochloride, and tetrodotoxin (TTX) were
obtained from Sigma (St. Louis, MO); Rp-8-Br-cGMPS and 8-pCPT-cGMP
were purchased from Calbiochem (La Jolla, CA). -Agatoxin TK was
obtained from Almone (Jerusalem, Israel).
Statistical Analysis. The data for each experiment were normalized relative to baseline and are presented as means ± S.E.M. n indicates the number of experiments. The significance of the difference between the mean was calculated by paired or unpaired Student's t test. Probability values (p) of less than 0.05 were considered to represent significant differences. Comparisons between control and experimental distributions of sEPSC amplitude and interevent intervals were made by performing Kolmogorov-Smirnov test. Distributions were considered different using a conservative critical probability level of p < 0.01.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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(n = 32), respectively, which are
comparable with the values reported previously
(Hwang and Dun, 1998). In all
experiments, neurons were clamped at -70 mV and EPSCs were evoked by
intra-RVLM stimulation with bipolar stimulating electrode every 20 s in the
presence of the GABAA receptor antagonist bicuculline methiodide
(10 µM) and the glycine receptor antagonist strychnine hydrochloride (0.5
µM).
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Exogenous NO-Induced Synaptic Potentiation. We examined initially the effect of L-arginine, the substrate for NO production by NOS, on the evoked EPSCs. In six of eight cells tested, bath application of 200 µM L-arginine for 10 min produced a significant enhancement of evoked EPSCs. The mean EPSC amplitude after L-arginine was increased by 24.9 ± 5.8% of the control baseline (n = 8; p < 0.05; paired Student's t test) (Fig. 2, A and C). The EPSCs recovered toward the control level within a few minutes after washout of L-arginine, suggesting the reversibility of L-arginine effect. At the end of each experiment, the non-NMDA receptor antagonist CNQX (20 µM) and NMDA receptor antagonist D-APV (50 µM) were added to the bath to ensure that the synaptic response was glutamatergic EPSC. In the same concentration, D-arginine did not cause a significant change of EPSCs (4.5 ± 2.3% of the control baseline, n = 4; p > 0.05; paired Student's t test) (Fig. 2C).
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To confirm that L-arginine-induced synaptic potentiation is caused by the synthesis of NO, we tested the effects of blocking nNOS. As shown in Fig. 2, B and C, the increase in the amplitude of EPSCs by L-arginine (200 µM) was completely prevented when 7-NI (100 µM), a selective nNOS inhibitor, was preperfused for more than 10 min before L-arginine application (n = 7; p < 0.05; unpaired Student's t test). In the presence of 7-NI, L-arginine increased the amplitude of EPSCs by a mean of 3.1 ± 1.7% of pre–L-arginine level. Additionally, bath application of 7-NI (100 µM) alone had a slight but not significant increase in the amplitude of EPSCs (7.5 ± 3.8% of the control baseline, n = 7; p > 0.05; paired Student's t test). These results suggest that the facilitating action of L-arginine on glutamatergic transmission on the RVLM neurons is mediated by the nNOS-mediated synthesis of NO.
Additional evidence that NO can induce a synaptic potentiation of glutamatergic transmission on the RVLM neurons came from experiments using the NO donors SIN-1 and spermine NONOate. Similar to L-arginine, bath application for 10 min of the SIN-1 (200 µM) or spermine NONOate (100 µM) produced a significant enhancement in six of eight and six of eight cells tested, respectively. The mean EPSC amplitude after SIN-1 or spermine NONOate was 24.9 ± 4.4 and 22.5 ± 4.3% of the control baseline, respectively (n = 8 and 8; p < 0.05; paired Student's t test) (Fig. 3).
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NO Enhances Synaptic Transmission through a sGC/cGMP/PKG-Coupled
Signaling Pathway. An established signal transduction pathway for NO is
activation of sGC, leading to an increase in intracellular cGMP levels
(Ignarro, 1991). Thus, to
address the involvement of cGMP in the action of NO on glutamatergic
transmission on the RVLM neurons, we investigated whether the pharmacological
inhibition of sGC was able to prevent the synaptic potentiation caused by
L-arginine and NO donors. Preincubation of the slices with 10 µM
ODQ, a selective inhibitor of sGC, caused no change in the amplitude of EPSCs
(107.6 ± 4.6% of the control baseline; n = 15; p >
0.05; paired Student's t test) but fully blocked the synaptic
potentiation caused by L-arginine, SIN-1, and spermine NONOate
(Fig. 4). In the presence of
ODQ (10 µM), bath application of L-arginine (200 µM), SIN-1
(200 µM), or spermine NONOate (100 µM) for 10 min produced only a minor
increase in the amplitude of EPSCs by 12.5 ± 3.2% (n = 5), 9.5
± 3.6% (n = 5), and 5.3 ± 3.5% (n = 5),
respectively, which were significantly different from those produced by
L-arginine, SIN-1, or spermine NONOate alone (p < 0.05;
unpaired Student's t test).
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Intracellular elevation of cGMP levels may result in the stimulation of PKG, which in turn modulates the function of a series of cellular substrates by increasing their phosphorylation state. To examine the role of PKG in the action of NO on glutamatergic transmission on the RVLM neurons, the effect of a selective PKG inhibitor, Rp-8-Br-cGMPS, on SIN-1- and spermine NONOate-induced synaptic potentiation was investigated. In these experiments, slices were incubated for at least 1 h in 50 µM Rp-8-Br-cGMPS before being transferred to the recording chamber, where the concentration was maintained at 10 µM. Pretreatment of the slices with Rp-8-Br-cGMPS completely blocked the SIN-1- and spermine NONOate-induced synaptic potentiation (Fig. 5, A and B). In the presence of Rp-8-Br-cGMPS, bath application of SIN-1 (200 µM) or spermine NONOate (100 µM) for 10 min produced only a minor increase in the EPSC amplitude by a mean of 6.5 ± 3.7 (n = 5) and 10.3 ± 5.9 (n = 5), respectively, which were significantly different from those produced by SIN-1 or spermine NONOate alone (p < 0.05; unpaired Student's t test). To further examine the role of cGMP in synaptic potentiation on the RVLM neurons, we examined the effect of a membrane-permeable cGMP analog, 8-pCPT-cGMP. As shown in Fig. 5C, in six of seven cells tested, bath application of 8-pCPT-cGMP (50 µM) for 20 min mimicked the facilitating action of L-arginine and NO donors. The mean EPSC amplitude after 8-pCPT-cGMP was increased by 24.5 ± 4.3% of the control baseline (n = 7; p < 0.05; paired Student's t test). These results suggest that NO-induced synaptic potentiation via a mechanism that is triggered by the activation of the sGC/cGMP/PKG-coupled signaling cascade.
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Presynaptic Expression of Spermine NONOate-Induced Synaptic
Potentiation. Because SIN-1 is now known to have other effects related to
its ability to produce peroxynitrite, we therefore used only spermine NONOate
to investigate the further mechanisms underlying the NO-induced synaptic
potentiation on the RVLM neurons (Holm et
al., 1998; Trackey et al.,
2001). To identify whether the NO action was on presynaptic or
postsynaptic sites, two different approaches were used. We initially addressed
this issue by examining the effect of spermine NONOate on the failure rate of
single-fiber EPSCs evoked by minimal stimulation, which reflects changes in
the presynaptic transmitter release
(Stevens and Wang, 1994;
Raastad, 1995). As a typical
example shown in Fig. 6A, the
expression of spermine NONOate-induced synaptic potentiation is accompanied by
a decrease in the synaptic failure rate. On average, the failure rate was
decreased from 62.4 ± 3.4% to 34.2 ± 2.6% after spermine NONOate
(100 µM) application (n = 5; p < 0.05; paired
Student's t test) (Fig.
6B).
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To further test the possibility that spermine NONOate-induced synaptic
potentiation through a presynaptic mechanism, we conducted studies examining
the effects of spermine NONOate on PPF. When the excitatory afferents to the
central neurons are activated twice with a short interval between each
stimulus, the response to the second stimulus is generally facilitated in
relation to the initial stimulus. This phenomenon is called a PPF and is
attributed to an increase the amount of transmitter release to the second
stimulus (Zucker, 1989). On
the other hand, the manipulations of presynaptic transmitter release may
result in the change in the magnitude of PPF. If the spermine NONOate-induced
synaptic potentiation involved a presynaptic mechanism of action, it will be
associated with a change in the magnitude of PPF. Alternatively, if spermine
NONOate induced synaptic modulation by another type of mechanism (e.g.,
reducing the sensitivity of postsynaptic receptors), then the PPF magnitude
should be relatively unaffected. To test this hypothesis, the magnitude of PPF
was determined at control before the application of spermine NONOate and 10
min after starting the application of spermine NONOate.
Figure 6C shows a typical
example of EPSCs synaptically evoked in response to a pair of stimuli with an
interpulse interval of 30 ms. We found that the increase of the amplitude of
EPSCs induced by spermine NONOate (100 µM) was accompanied by a decrease in
the magnitude of PPF. The magnitude of PPF was 2.29 ± 0.18% before and
1.67 ± 0.11% (n = 5; p < 0.05; paired Student's
t test) during the application of spermine NONOate. These results
suggest that NO may act presynaptic site to modulate the transmitter release
mechanisms in the RVLM.
Effects of NO on Spontaneous Excitatory Postsynaptic Currents. To further confirm the possibility that NO modulates the evoked EPSCs through a presynaptic mechanism, we examined the effects of spermine NONOate on sEPSCs. sEPSCs in the RVLM neurons were measured under voltage clamp at -70 mV and were pharmacologically isolated from spontaneous inhibitory currents by the inclusion of 10 µM bicuculline methiodide and 0.5 µM strychnine hydrochloride in the ACSF perfusing the slices. The sEPSCs were totally blocked by bath coapplication of CNQX (20 µM) and D-APV (50 µM), confirming them to be true glutamate receptor-mediated events. Under control conditions, sEPSCs had a mean amplitude of 6.92 ± 0.33 pA and a variable frequency ranging from 1.0 to 1.6 Hz (mean, 1.32 ± 0.12 Hz; n = 5). In five cells tested, spermine NONOate (100 µM) markedly increased the mean frequency of the sEPSCs from 1.32 ± 0.12 to 2.53 ± 0.28 Hz (p < 0.05; paired Student's t test) (Fig. 7, A and E). Significant differences in cumulative interevent interval distributions were observed in all five cells tested during spermine NONOate application; i.e., spermine NONOate shifted the interevent interval distribution of sEPSCs to shorter intervals (p < 0.001; Kolmogorov-Smirnov test). A typical example of recorded cell is shown in Fig. 7C. However, there was no significant effect of spermine NONOate (100 µM) on the sEPSC amplitude. This can be observed by a lack of effect of spermine NONOate on either the amplitude histogram (Fig. 7B) or the cumulative probability plots (Fig. 7B, inset; p = 0.54; Kolmogorov-Smirnov test). The mean amplitude of sEPSCs recorded in the presence of spermine NONOate (100 µM) was 8.08 ± 0.56 pA, which was of comparable amplitude with that of sEPSCs recorded under control conditions (6.92 ± 0.33 pA; p > 0.05; paired Student's t test).
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The spontaneous synaptic events recorded from the RVLM neurons could be roughly divided into two components: TTX-sensitive, action potential-dependent sEPSCs and TTX-resistant, action potential-independent miniature EPSCs (mEPSCs). The action potential-dependent sEPSCs arise from presynaptic impulses, whereas the action potential-independent mEPSCs are thought to result from spontaneous fusion of neurotransmitter-containing vesicles to the presynaptic terminal membrane in a manner independent of the activation of presynaptic voltage-dependent ion channels. We next examined the effect of spermine NONOate on mEPSCs to determine whether NO can modulate action potential-independent spontaneous events. TTX (1 µM) was added to the perfusate in the presence of bicuculline methiodide and strychnine hydrochloride to eliminate sEPSCs arising from presynaptic impulses. In all cells recorded, the efficacy of TTX block of Na+ channels was monitored by observing the disappearance of the evoked EPSCs during maximal electrical stimulation (data not shown). Application of TTX (1 µM) alone reduced both the amplitude and frequency of sEPSCs. Amplitude histograms show that TTX caused a reduction in the relative frequency of large-amplitude synaptic events. Additionally, TTX also reduce the relative frequency of large-amplitude synaptic events compared with control base (data not shown). Figure 8 illustrates that application of spermine NONOate (100 µM) also led to a significant increase in the frequency of mEPSCs. In five cells tested, spermine NONOate (100 µM) markedly increased the mean frequency of the sEPSCs from 0.62 ± 0.14 to 1.08 ± 0.15 Hz (p < 0.05; paired Student's t test) (Fig. 8, A and E). Significant differences in cumulative interevent interval distributions were observed in all five cells tested during spermine NONOate application; i.e., spermine NONOate shifted the interevent interval distribution of mEPSCs to shorter intervals (p < 0.01; Kolmogorov-Smirnov test). A typical example of recorded cell is shown in Fig. 8C. However, there was no significant effect of spermine NONOate (100 µM) on the mEPSC amplitude. This can be observed by a lack of effect of spermine NONOate on either the amplitude histogram (Fig. 8B) or the cumulative probability plots (Fig. 8B, inset; p = 0.94; Kolmogorov-Smirnov test). The mean amplitude of mEPSCs recorded in the presence of spermine NONOate (100 µM) was 6.63 ± 0.35 pA, which was of an amplitude comparable with that of mEPSCs recorded under control condition (6.19 ± 0.28 pA; p > 0.05; paired Student's t test). Therefore, these data further suggest that NO may act presynaptically to enhance the amount of glutamate release without changing the postsynaptic sensitivity to glutamate.
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N-Type Ca2+ Channels Contribute to the Spermine
NONOate-Induced Synaptic Potentiation. In a final series of experiments,
we examined the possibility that NO modulates evoked EPSCs through an action
on presynaptic voltage-sensitive Ca2+ channels, which
contribute to supporting glutamate release. If spermine NONOate acts on
presynaptic Ca2+ channels to affect glutamate release
mechanisms, it would do so through one or more channel subtypes. We therefore
examined the effect of spermine NONOate on the amplitude of EPSC before and
after selective blockade of each of Ca2+ channel
subtypes. We first examined the possible contribution of N-type
Ca2+ channel enhancement to spermine NONOate-induced
synaptic potentiation. A representative cell recorded under this condition is
shown in Fig. 9A. Application
of -conotoxin-GVIA 1 µM, a concentration that should selectively
block N-type Ca2+ channels
(Kasai et al., 1987), caused a
rapid, robust, and irreversible suppression of the amplitude of EPSCs by 72%
and completely blocked the spermine NONOate action. In six neurons tested,
spermine NONOate (100 µM) produced a 5.2 ± 3.6% increase in the
amplitude of EPSCs after the application of -conotoxin-GVIA, which was
significantly different from that produced by spermine NONOate alone (22.5
± 4.3%, n = 8; p < 0.05; unpaired Student's
t test) (Fig. 9E).
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We next investigated the role of P/Q-type Ca2+
channels in the modulation caused by spermine NONOate. -Aga-TK, a toxin
purified from the venom of funnel web spider, was used in these experiments.
-Aga-TK has been reported to selectively block the P-type
Ca2+ channel at nanomolar concentrations, whereas at
concentration >100 nM, it blocks not only P-type but also Q-type
Ca2+ channels
(Wheeler et al., 1994). As a
representative experiment shown in Fig.
9B, application of 200 nM -Aga-TK reduced the amplitude of
EPSCs by 32% compared with baseline. After the application of -Aga-TK,
bath application of spermine NONOate (100 µM) for 10 min was still able to
potentiate the residual EPSC amplitude by 21.8 ± 5.2% compared with
baseline (n = 5), which was not significantly different from the
synaptic potentiation produced by spermine NONOate alone (22.5 ± 4.3%,
n = 8; p > 0.05; unpaired Student's t test)
(Fig. 9E).
We further examined the possible contribution of L-type Ca2+ channel enhancement to spermine NONOate-induced synaptic potentiation. As a representative experiment shown in Fig. 9C, pretreatment of the slices with the selective L-type Ca2+ channel blocker nimodipine (20 µM) affected neither the basal EPSC amplitude nor the spermine NONOate-induced synaptic potentiation. In five neurons tested, spermine NONOate (100 µM) produced a 26.7 ± 4.8% increase in the amplitude of EPSCs after the application of nimodipine, which was not significantly different from that produced by spermine NONOate alone (22.5 ± 4.3%, n = 8; p > 0.05; unpaired Student's t test) (Fig. 9E). The conclusion from this series of experiments is that spermine NONOate-induced enhancement of glutamatergic synaptic transmission on the RVLM neurons is most likely through an increase the activity of presynaptic N-type but not P/Q-type or L-type Ca2+ channel subtypes.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Concerning the locus of NO-induced synaptic potentiation, a presynaptic
site of action seems to be involved. We can give three lines of evidence to
support this conclusion. First, the NO donor spermine NONOate produces a
significant decrease in the failure rate of single-fiber EPSCs evoked by
minimal stimulation (Fig. 6, A and
B). Second, an increase in synaptic transmission by spermine
NONOate was accompanied by a decrease in the magnitude of PPF ratio of
synaptically evoked responses (Fig.
6C), which is usually considered an indication of a presynaptic
mode of drug actions (Zucker,
1989). Third, and most importantly, spermine NONOate significantly
increases the frequency of both sEPSCs and mEPSCs but did not affect their
amplitude (Figs. 7 and
8). A change in the amplitude
of sEPSCs and mEPSCs is usually interpreted as a postsynaptic modification,
whereas a change in their frequency is typically associated with mechanisms
that increase the probability of nerve-evoked transmitter release
(Katz, 1969). Thus, the lack
of effect of spermine NONOate on the amplitude of sEPSCs and mEPSCs also
implies that the modulation of synaptic transmission by NO on the RVLM neurons
is not mediated by a change in postsynaptic sensitivity to glutamate.
NO has been shown to activate several signal transduction pathways,
including sGC, to increase cGMP levels
(Garthwaite, 1991). The
involvement of NO/cGMP pathway in the modulation of glutamatergic transmission
has been demonstrated in slice preparations from the hippocampus
(O'Dell et al., 1991;
Boulton et al., 1994) and locus
ceruleus (Pineda et al., 1996)
and in cultured hippocampal neurons
(Arancio et al., 1995). In the
present results, both L-arginine and NO donor-induced synaptic
potentiation were prevented by a selective NO-sensitive sGC inhibitor, ODQ
(Fig. 4). It is likely that NO
is acting through a NO-sensitive sGC/cGMP signaling pathway to increase
glutamatergic transmission on the RVLM neurons. Various potential mechanisms
can account for the cellular function of cGMP in different systems. The
specific targets for cGMP are PKG
(Paupardin-Tritsch et al.,
1986), cyclic nucleotide-gated cationic channels
(Fesenko et al., 1985), and
cyclic nucleotide-stimulated and -inhibited phosphodiesterases
(Nicholson et al., 1991). In
the present study, experiments were performed with activator (8-pCPT-cGMP) or
inhibitor (Rp-8-Br-cGMPS) of PKG to characterize the pathway involved
in NO effect. The experiments showed that preincubation of the slices with
Rp-8-Br-cGMPS fully prevented SIN-1- and spermine NONOate-induced
synaptic potentiation (Fig. 5, A and
B), whereas bath application of 8-pCPT-cGMP mimicked the action of
NO donors (Fig. 5C). In
addition, both 8-pCPT-cGMP and Rp-8-Br-cGMPS have a negligible
activity on cyclic nucleotide-stimulated and -inhibited phosphodiesterases,
indicating that the activation of cyclic-nucleotide phosphodiesterases is not
necessary for the NO-induced synaptic potentiation. Therefore, our results
support the idea that NO-induced synaptic potentiation on the RVLM neurons is
mediated via a cGMP/PKG-coupled signaling pathway. However, we could not
exclude the possibility that the increase in cGMP levels by NO-stimulated sGC
may directly modulate presynaptic cyclic nucleotidegated cationic channels,
which leads to depolarization of the presynaptic terminals; potentiation of
transmitter release may also play a part role in the NO-induced synaptic
potentiation (Ludwig et al.,
1998).
What is the biological step downstream of cGMP/PKG responsible for the
NO-induced synaptic potentiation? The most likely candidate is their
facilitatory coupling with presynaptic voltage-sensitive
Ca2+ channels, which in turn enhances glutamate release.
Various subtypes of voltage-sensitive Ca2+ channels are
known to be involved in neurotransmitter release. In the present study, by
using the highly selective inhibitors of voltage-sensitive
Ca2+ channels, distinct subtypes of voltage-sensitive
Ca2+ channels contributing to the evoked release of
glutamate on the RVLM neurons were identified. Data reported in this article
represent the first experimental evidence that N- and P/Q-type
Ca2+ channels make the major contribution to the evoked
glutamate release in the RVLM area (Fig.
9D). The finding that spermine NONOate-induced synaptic
potentiation dropped from 22 to 5% after exposure to the N-type
Ca2+ channel blocker -CgTX GVIA
(Fig. 9, A and D) is evidence
that the enhancement of this channel activity is the primary mechanism of
NO-induced potentiation of glutamate release from presynaptic nerve terminals.
In salamander retinal ganglion cells, a recent study also demonstrated that NO
can enhance N-type Ca2+ channel activity by facilitating
their voltage-dependent activation via a mechanism involving
sGC/cGMP/PKG-dependent phosphorylation
(Hirooka et al., 2000). The
evidence that blockade of P/Q-type and L-type Ca2+
channels had no effect on the spermine NONOate-induced synaptic potentiation
seems to rule out the involvement of these channel subtypes.
Our observation that spermine NONOate also enhances the frequency but not
the amplitude of the mEPSCs suggests that NO has effect in addition to its
modulation of voltage-sensitive Ca2+ channels. Because
concentrations of Ca2+ channel blockers sufficient to
abolish all evoked transmitter release have no effect on mEPSC frequency,
changes in mEPSC frequency are not likely to be the result of changes in
Ca2+ influx from voltage-sensitive
Ca2+ channels at the resting membrane potentials
(Scanziani et al., 1992). The
NO-mediated increase in mEPSC frequency therefore may reflect a direct action
on the exocytotic apparatus or protein kinase-dependent phosphorylation of
targets that regulate the vesicle release machinery.
An important issue that arose in this study was whether the NO
concentrations released from donor compounds could be reached under
physiological or pathological conditions in the brain. Although the local NO
concentrations in slice preparations produced by SIN-1 (200 µM) and
spermine NONOate (100 µM) remain unknown, it has been demonstrated that
spermine NONOate at a concentration of 82.7 µM will generate a peak NO
concentration in the Tyrode's solution of 3 to 4 µM
(Ramamurthi and Lewis, 1997).
Moreover, tissue-derived factors may accelerate NO breakdown in slice studies,
because NO decays more quickly when perfused over tissues compared with the
experiments in tubes (Palmer et al.,
1987). In a recent report, it was calculated that the
concentrations of NO produced by another NO donor,
2,2-diethyl-1-nitroso-oxyhydrazine (100–300 µM), were in the 100 nM
range in the hippocampal slices (Bon and
Garthwaite, 2001). Hence, the local NO concentrations produced by
100 µM spermine NONOate in the present study should be in the 50 to 100 nM
range. Because it is known that the physiological range of NO concentration in
the brain is 10 to 100 nM and ischemic conditions produce NO of about 2 to 4
µM (Shibuki and Okada,
1991; Malinski and Taha,
1992), the NO-induced synaptic potentiation on the RVLM neurons
described in the present study would be expected to occur in vivo under
physiological or ischemic situations.
In conclusion, we here extended the earlier studies on the role of NO and
glutamate in the RVLM, providing further evidence that NO may substantially
enhance glutamatergic transmission on the RVLM neurons. Consisting of premotor
sympathetic neurons, the RVLM is believed to maintain basal levels of systemic
arterial pressure (Ross et al.,
1984) by providing tonic excitation to preganglionic sympathetic
neurons in the intermediolateral cell column of the spinal cord. The
glutamatergic afferents to the RVLM control the premotor neurons, which in
turn excite the preganglionic neurons in the spinal cord, which send their
axons to the periphery and synapses with sympathetic postganglionic neurons.
Via the increase in the excitatory synaptic transmission in the RVLM, NO could
indirectly excite preganglionic neurons of the spinal cord, increasing their
firing rate, and trigger sympathoexcitatory functions, such as increasing SAP
and heart rate.
|
Footnotes |
|---|
ABBREVIATIONS: RVLM, rostral ventrolateral medulla; NO, nitric
oxide; NOS, nitric-oxide synthase; iNOSg, inducible nitric-oxide synthase;
nNOS, neuronal nitric-oxide synthase; sGC, soluble guanylyl cyclase; PKG,
cGMP-dependent protein kinase; ACSF, artificial cerebrospinal fluid; EPSC,
excitatory postsynaptic current; sEPSC, spontaneous excitatory postsynaptic
current; QX-314, lidocaine N-ethyl bromide; mEPSC, miniature
excitatory postsynaptic current; 7-NI, 7-nitroindazole; ODQ,
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; SIN-1,
3-morpholinylsydnoneimine; CNQX, 6-cyano-7-notroquinoxaline-2,3-dione;
spermine NONOate,
N-[4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]-butyl]-1,3-propanediamine;
D-APV, D-(-)--amino-5-phosphonopentanoic acid;
TTX, tetrodotoxin; Rp-8-Br-cGMPS, Rp-8-bromo-guanosine
3',5'-cyclic monophosphorothioate; 8-pCPT-cGMP,
8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate; PPF,
paired-pulse facilitation; -Aga-TK, -agatoxin-TK.
Address correspondence to: Dr. Kuei-Sen Hsu, Department of Pharmacology, College of Medicine, National Cheng Kung University, 1, Ta-Hsiue Rd., Tainan 701, Taiwan. E-mail: richard{at}mail.ncku.edu.tw
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72% of the EPSCs. It is clear that spermine NONOate
(100 µM) failed to affect the fraction of synaptic responses insensitive to