Actions of reactive oxygen species on AH/type
2 myenteric neurons in guinea pig distal colon
S.
Wada-Takahashi and
K.
Tamura
Department of Physiology, Kanagawa Dental College, Yokosuka
238-8580, Japan
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ABSTRACT |
With conventional intracellular
recording methods, we investigated the mechanism of actions of reactive
oxygen species (ROS) derived from hypoxanthine and xanthine oxidase
(HX/XO) reactions on AH/type 2 myenteric neurons in the guinea pig
distal colon. Of the 54 neurons to which HX/XO was applied, 32 neurons
showed a transient membrane hyperpolarization(s) followed by a
long-lasting membrane depolarization. Two additional groups of 10 myenteric neurons exhibited only a membrane hyperpolarization(s) or a
late-onset membrane depolarization, respectively, and the remaining two
neurons did not show any response to HX/XO. Analysis of changes of the input resistance induced by HX/XO indicated that suppression and augmentation of the conductance of Ca2+-dependent
K+ channels are the ionic mechanisms underlying the
membrane hyperpolarization and depolarization, respectively. The
effects of HX/XO on myenteric neurons were mimicked by application of
caffeine or H2O2. The results suggest that
OH·, but neither H2O2 nor
O2·
, is responsible for HX/XO-induced
responses. The intracellular Ca2+ store may be the acting
site of ROS in colonic AH/type 2 neurons.
superoxide anion radical; hydrogen peroxide; hydroxyl radical; AH/type 2 neuron
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INTRODUCTION |
REACTIVE OXYGEN
SPECIES (ROS), including superoxide anion radical
(O2·
) and its metabolic intermediates,
H2O2 and hydroxyl radical (OH·),
are highly reactive substances produced by a variety of reactions in
many biological systems (1, 6, 10). Under physiological conditions, ROS are continuously produced in small amounts as metabolic
byproducts in virtually all living cells. The electron transport chain
associated with the mitochondrial membranes produces ROS during the
univalent process of reduction of O2 to H2O.
Endogenous enzymes such as superoxide dismutase (SOD), catalase (CAT),
and glutathione peroxidase decompose or inactivate these naturally arising ROS to protect the cell from oxidative damage (5,
35).
In various pathological states, however, ROS are often produced at
amounts surpassing these physiological defense mechanisms, triggering a
chain reaction that leads to serious tissue damage. In the central
nervous system, overproduction of mitochondria-derived ROS by the
impaired electron transport chain has been suggested to contribute to
Alzheimer's and Parkinson diseases (5). Transgenic mice
with the mutant form of SOD produce excessive amounts of OH· and develop degenerative changes in the spinal cord
and brain, as found in amyotrophic lateral sclerosis (18).
When a tissue is exposed to hypoxia, xanthine dehydrogenase in the
vascular endothelial cells is converted to xanthine oxidase (XO), which in turn produces O2·
after
reoxygenation of the tissue (1, 6, 10, 15). The tissue
damage by hypoxia-reoxygenation- or
ischemia-reperfusion-derived O2·
and
its metabolites is referred to as reperfusion injury and has been
implicated in the pathogenesis of various diseases or conditions such
as myocardial infarction (1, 14), mucosal ulceration in
the gastrointestinal tract (31), and dysfunction of
transplanted organs (26). In sites with inflammation,
neutrophils recruited from the blood circulation are activated and
secrete ROS produced by the action of NADPH oxidase (9).
Tissue injury and subsequent vascular dysfunction by neutrophil- or
macrophage-derived ROS have been implicated in diseases such as
rheumatoid arthritis, atherosclerosis, and the idiopathic inflammatory
bowel diseases (IBD) including Crohn's disease and ulcerative colitis
(9, 10, 35).
On the other hand, accumulating evidence now indicates that ROS might
be serving as signaling molecules at subtoxic concentrations in various
biological processes (14, 25). In the nervous system, it
has been shown that ROS regulate the release of neurotransmitters in
the peripheral and central nervous systems. In the rat hippocampus, release of endogenous amino acids is enhanced by ROS (22).
Depolarization-induced Ca2+ uptake and release of GABA from
the cerebral cortex synaptosomes are strongly inhibited by antioxidant
chemicals (37). The release of norepinephrine from the
sympathetic nerve terminals in the guinea pig intestine is constantly
facilitated by O2·
and its metabolites
(12). In addition, the kinetics of the inward rectifier
K+ channels is modulated by ROS (8, 11). The
reversible effects of ROS shown in these studies are indicative of the
possible role of ROS as neuromodulators in the nervous system.
In the present study, therefore, we examined the effects of ROS on the
colonic myenteric neurons, in view of the importance of ROS as possible
signaling molecules in the enteric circuits under physiological as well
as pathophysiological conditions. The gastrointestinal tract undergoes
physiological cycles of anemia and hyperemia during the normal
digestive activities, and the enteric neurons might be constantly
exposed to cyclical assaults of local hypoxia or oxidative stress. In
the IBD, extravasation and infiltration of large numbers of
inflammatory leukocytes into the colonic interstitium have been
indicated in the active region (10). Therefore, ROS
secreted from activated leukocytes might be acting on the colonic
enteric neurons, causing functional disorders in colonic motility such
as diarrhea and constipation in patients with IBD. Preliminary results
of this study have been published in abstract form (30).
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METHODS |
Seventy-four adult male guinea pigs (250-350 g) were used
in this study. The segments of distal colon were obtained after stunning by a sharp blow to the head and exsanguination. This method of
death was approved by the animal experimentation committee of Kanagawa
Dental College. Flat sheet preparations of colonic longitudinal muscle
with adherent myenteric ganglia were obtained by removing the mucosal
and circular muscle layers. The preparations were then mounted in a
small recording chamber and superfused with normal Krebs solution
gassed with 95% O2-5% CO2 and warmed to
37°C. Composition of the Krebs solution was (in mM) 120.9 NaCl, 5.9 KCl, 1.2 MgCl2, 14.4 NaHCO3, 1.33 NaH2PO4, 2.5 CaCl2, and 11.5 glucose. Myenteric ganglia were viewed under an inverted microscope
(TE300; Nikon, Tokyo, Japan) equipped with Hoffman modulation contrast
optics. Nifedipine (1 µM) and atropine (1 µM) were added to the
Krebs solution to immobilize the movement of the tissue by contractions
of the longitudinal muscles.
Transmembrane potentials of myenteric neurons were measured with
conventional intracellular recording methods using 2 M KCl-filled microelectrodes (70-120 M
). Action potentials were evoked by intrasomatic injection of depolarizing current (0.1-1.0 nA, 200- to 500-ms square pulses) through the microelectrode and the bridge circuitry contained in the amplifier (CEZ-3100; Nihon Koden, Tokyo, Japan). Action potentials were also evoked by focal electrical stimulation of interganglionic fiber tracts with Teflon-coated platinum
electrodes (diameter 20 µm), and somatic spikes were triggered by
antidromic invasion from the activated neurites. The input resistance
of the impaled neurons was calculated from current-voltage
relationships produced by three or four different steps of
hyperpolarizing currents from a step pulse generator (SET-1201, Nihon
Koden). The records were reproduced on a thermal array recorder
(RTA-1100M, Nihon Koden) or on a digital storage oscilloscope (CS-8010;
Kenwood, Tokyo, Japan) from original records on digital audiotapes.
Amplitudes and durations at half-amplitude of the action
potentials were directly measured from the oscilloscope. Data are
expressed as means ± SD or as means ± SE.
The chemical agents used in this study were hypoxanthine (HX), SOD,
CAT, and caffeine (Sigma Chemical, St. Louis, MO). TTX, atropine
sulfate monohydrate, nifedipine chloride, H2O2,
and DMSO were obtained from Wako Chemicals (Tokyo, Japan). XO was
purchased from Roche (Basel, Switzerland). HX and XO were added
together (HX/XO) in the superfusing Krebs solution as a donor of
O2·
, and other chemicals were also
applied by addition to the Krebs solution. In some experiments,
H2O2 (15 mM) was applied from a fine-tipped
pipette positioned in the close vicinity (50-100 µm) of the
impaled neurons by pressure injection with nitrogen pulses of
controlled duration and pressure.
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RESULTS |
Electrophysiological properties of colonic myenteric neurons.
Successful intracellular recordings were made from 110 colonic
myenteric neurons with AH/type 2 electrophysiology. The membrane potential of these neurons ranged from
41 to
80 mV, and all of them
had large action potentials (>68 mV). Stable intracellular recordings
were maintained in these neurons for periods of 20 min to several
hours. They were identified as AH/type 2 neurons on the basis of the
electrophysiological characteristics of this type of myenteric neurons
reported in earlier studies (27, 33, 34). These
characteristics of AH/type 2 neurons are 1) discharge of one
or two action potentials only at the onset of intracellular injection
of depolarizing current pulses, 2) broad action potentials with a half-duration >1.0 ms, and 3) after-spike
hyperpolarizations (AH) >2 s in duration. Other electrophysiological
properties including the resting membrane potential, the input
resistance, and the amplitude of action potentials of AH/type 2 neurons
recorded in this study were compatible with those reported in a
previous study on the myenteric neurons in this region (Table
1; Ref. 33).
Effects of HX/XO on membrane excitability.
Effects of ROS derived from HX/XO reaction were examined in 54 AH/type
2 neurons. When HX (5-100 µM) alone was added in the superfusing
Krebs solution, no detectable changes in the membrane potential and the
input resistance were observed in any of eight neurons tested. In 32 of
54 neurons (59.3%), application of HX/XO produced a characteristic
biphasic response consisting of a transient membrane hyperpolarization
accompanied by a large decrease in the input resistance and a
subsequent long-lasting membrane depolarization that occurred with an
increase in the input resistance (Fig.
1). The HX/XO-induced transient membrane
hyperpolarization began within 60-90 s after application of
HX/XO-containing Krebs solution and lasted for a short period of
20-60 s (Fig. 1A). The subsequent long-lasting membrane
depolarization began to develop immediately after the transient
hyperpolarization, reaching a peak in 2-5 min, and often outlasted
the superfusion of HX/XO-containing Krebs solution. In 17 of 32 neurons
that exhibited a biphasic response, another membrane hyperpolarization
followed the slow membrane depolarization after washout of HX/XO (Fig.
1B). Of the remaining 22 neurons, 10 neurons (18.5%) showed
only a membrane hyperpolarization(s) to HX/XO application, whereas
another 10 neurons (18.5%) responded with a slow membrane
depolarization without preceding transient membrane hyperpolarization
(Fig. 2A). Two neurons (3.7%)
did not show any responses to HX/XO application (Table
2). In 20 of 42 neurons that exhibited
membrane hyperpolarization to HX/XO, two or three transient
hyperpolarizations occurred cyclically with short intervals (10-15
s) before development of the slow depolarization (Fig.
3A).

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Fig. 1.
Transient membrane hyperpolarizing and slow
depolarization evoked in an AH/type 2 colonic neuron by superfusion of
HX/XO-containing Krebs solution. A: hypoxanthine (HX; 10 µM) + xanthine oxidase (XO; 0.01 U/ml) application induced a
short-lasting membrane hyperpolarization accompanied by a large
decrease in the input resistance. A somatic action potential evoked by
stimulation of the fiber tract was suppressed during the
hyperpolarization (arrow). B: a long-lasting membrane
depolarization slowly developed after the initial hyperpolarization,
triggering action potentials. Somatic action potential was evoked by
stimulating the fiber tract at intervals of 30 s throughout the
experiment. The resting membrane potential of this neuron before HX/XO
application was 65 mV. A and B are continuous
recordings from the same neuron. Top traces, transmembrane
potential; bottom traces, injected current.
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Fig. 2.
Effects of superoxide dismutase (SOD) and catalase (CAT)
on HX/XO-induced slow membrane depolarization in an AH/type 2 neuron.
A: application of HX (10 µM) and XO (0.01 U/ml) induced a
slowly developing membrane depolarization accompanied by a large
increase in the input resistance and discharge of action potentials.
B: addition of SOD (38 U/ml) in HX/XO-containing solution
did not suppress the slow membrane depolarization but enhanced the
membrane excitability, which was reflected by increased spike
discharge. C: CAT (5 U/ml) incompletely delayed the onset of
the HX/XO-induced membrane depolarization and suppressed discharge of
action potentials. Somatic action potential was evoked by stimulating
the fiber tract at intervals of 30 s throughout the experiment.
Top traces, transmembrane potential; bottom
traces, injected current.
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Fig. 3.
Reversal potential for the hyperpolarizing response
produced by HX (10 µM) and XO (0.01 U/ml) application in an AH/type 2 neuron. A: the amplitude of transient membrane
hyperpolarization was increased when the membrane potential was clamped
to 30 mV and was offset or nullified when the membrane
potential was clamped to below 90 mV. B: relation
between the membrane potential and the maximal amplitude of the
membrane hyperpolarization. Extrapolation of the line crosses the
abscissa at 100 mV and near the estimated K+ equilibrium
potential (n = 3, means ± SD).
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Complete recovery of neurons from these effects to control levels often
required 15-60 min after washout of the HX/XO-containing solution.
There was no relation between the type of response induced by HX/XO and
the electrophysiological properties of the impaled neurons such as the
resting membrane potential, amplitude of the action potential, and
amplitude and duration of the AH. Neither membrane hyperpolarization
nor depolarization was affected by addition of 0.2 µM TTX
(n = 5) in the HX/XO-containing Krebs solution, suggesting that these effects were direct actions on the postsynaptic membrane of the impaled neurons.
To assess the ionic mechanism underlying the transient
hyperpolarization induced by HX/XO, the membrane potentials were
current-clamped to various potentials from
30 to
120 mV
(n = 3). In all three neurons, the amplitude of
hyperpolarization was increased at more positive potentials than the
resting membrane potential and was decreased or nullified when clamped
to between
90 and
120 mV (Fig. 3A). The expected
reversal potential for the response appeared to be from approximately
90 to
100 mV, near the estimated K+ equilibrium
potential of the myenteric neurons (Fig. 3B).
Current-voltage slopes obtained using step-pulse methods during the
membrane hyperpolarization and depolarization intersected the control
slope before HX/XO application between
90 and
100 mV (Fig.
4). These observations suggested that
K+ conductance was enhanced during the HX/XO-induced
membrane hyperpolarization and attenuated during the slow
depolarization.

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Fig. 4.
Current-voltage relation slopes obtained during the
maximal hyperpolarization ( ) and the maximal
depolarization ( ) produced by HX (10 µM) and XO (0.01 U/ml) application. Both slopes intersect the control slope obtained
before HX/XO application ( ) between 90 and 96 mV
(broken lines), an estimated K+ equilibrium potential for
myenteric neurons. Data points are changes in the membrane potential to
steps of hyperpolarizing currents and were obtained from an experiment
shown in Fig. 8.
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The amplitude and duration of the transient membrane hyperpolarization
and slow depolarization induced by HX/XO varied significantly from cell
to cell even in the same neuron, depending on the state of membrane
excitability of each neuron before HX/XO application. The maximal
amplitudes of hyperpolarization ranged from
1.6 to
18.4 mV, and the
maximal depolarizations ranged from +4.8 to +15.6 mV. The average
duration of transient membrane hyperpolarization during the biphasic
response induced by 10 µM HX and 0.01 U/ml XO was 38.7 ± 16.8 s (n = 6) and that of the slow depolarization was 293.7 ± 153.9 s (n = 6). The analysis of the
input resistance during the HX/XO-induced membrane hyperpolarization
revealed a positive dose-dependence between the reduction of the input
resistance and the concentrations of HX over the range 10 nM to 100 µM (Fig. 5). No correlation, however,
was found between the increase of the input resistance during the
depolarization and HX concentration (Fig. 5), suggesting the
possibility that the mechanisms underlying the generation of membrane
hyperpolarization and depolarization by HX/XO might not be identical.

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Fig. 5.
Dose-response relations for changes in the input
resistance during HX/XO application. Left ordinate gives the maximal
decrease in the input resistance during hyperpolarization
( ), and right ordinate gives the increase of input
resistance during the slow membrane depolarization normalized to the
control ( ). Both values are expressed as a percentage
of the control. Abscissa gives the concentration of HX
(n = 3, mean ± SE).
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Suppression of action potentials.
When the action potentials of AH/type 2 neurons were evoked either by
focal stimulation of the fiber tract or by intracellular injection of
depolarizing current pulses, HX/XO reversibly suppressed the generation
of somatic action potentials. During transient membrane
hyperpolarization the onset of somatic action potentials triggered by
antidromic invasion of the initial segment spike was often delayed, and
they were completely abolished in six neurons (Fig. 1A). The
time delay from fiber tract stimulation to appearance of the remaining
initial segment spike in the soma, however, was unaffected, suggesting
that voltage-dependent Na+ channels in the long process or
neurite were not susceptible to HX/XO.
Na+ and Ca2+ carry the inward currents of
action potentials of myenteric AH/type 2 neurons, and Ca2+
remains as the carrier of inward current during blockade of
Na+ channels with TTX (34). To investigate the
mechanism underlying the inhibitory effect of HX/XO on the somatic
action potentials, Ca2+ spikes of AH/type 2 neurons were
evoked by injection of depolarizing current pulses with TTX (0.2 µM)
in the superfusing solution (n = 4). Treatment of
action potentials with TTX reduced the amplitude and rate of rise
but did not abolish the action potentials (Fig. 6, A and B).
Exposure to HX/XO in the superfusing solution containing TTX
significantly delayed the onset (Fig. 6C) and reduced the amplitude of action potentials afterwards in each of four neurons (Fig.
6D). The inhibitory effect of HX/XO on the somatic action potential was reversed after washout of HX/XO-containing solution for
15-30 min (Fig. 6E). The reduction of the amplitude of
Ca2+ action potentials was observed throughout the period
of HX/XO superfusion regardless of the responses occurring in the
membrane potential, indicating its direct effect on the
Ca2+ currents.

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Fig. 6.
Suppression of Ca2+ action potentials by
HX/XO. A: a somatic action potential evoked by injection of
depolarizing current pulse in an AH/type 2 neuron. B:
addition of TTX (0.2 µM) in the superfusing solution decreased the
amplitude of the action potential but did not abolish it. Application
of HX (10 µM) and XO (0.01 U/ml) in the presence of TTX delayed the
onset of Ca2+ action potential (C) and reduced
the amplitude (D). E: the amplitude of
Ca2+ action potential was completely restored after washout
of HX/XO by TTX-containing Krebs solution for 15 min. Top
traces, transmembrane potential; bottom traces,
injected current.
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Effects on AH.
During the HX/XO-induced membrane hyperpolarization, the size of AH
that followed action potentials evoked by the fiber tract stimulation
was significantly increased, whereas the AH were suppressed during the depolarization (Fig. 7). The
average amplitude of the AH was increased during the hyperpolarization
to 170.9 ± 11.5% (n = 5) of control amplitude
before HX/XO application, whereas the duration of AH was increased to
221.7 ± 32.6% (n = 5) of control duration. On
the other hand, these values decreased during the slow depolarization
to 38.8 ± 10.5% (n = 5) of control amplitude and
15.1 ± 6.8% (n = 5) of control duration,
respectively. The hyperpolarizing undershoot of the action potentials,
however, was not affected regardless of the changes occurring in the
membrane potential of the impaled neurons (Fig. 7). These observations suggest that HX/XO-derived ROS strongly enhance and attenuate the
conductance of the Ca2+-dependent K+ channels
without affecting the kinetics of the delayed-rectifier K+
channels that form the hyperpolarizing undershoot of the action potentials (28, 36).

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Fig. 7.
Effects of HX/XO on hyperpolarizing undershoot and AH
evoked in an AH/type 2 neuron. A: hyperpolarizing
undershoots on a fast time base (left) and after-spike
hyperpolarizations (AH) on a slow time base (right) that
followed action potentials evoked by fiber tract stimulation before
HX/XO application. B: during the membrane hyperpolarization
induced by HX (10 µM) and XO (0.01 U/ml) application, the amplitude
and duration of hyperpolarizing undershoot were not changed
(left), but the size of AH was significantly enhanced
(right). C: during the membrane depolarization
that occurred 5 min after HX/XO application, hyperpolarizing overshoot
was not affected (left), but AH was significantly suppressed
(right).
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In some neurons, however, the enhancement and reduction of AH amplitude
did not occur in parallel with the prolongation and shortening of AH
duration (Fig. 1). This phenomenon appeared to be the result of changes
in the electrical gradient for K+ caused by large movements
of the membrane potential toward and away from the K+
equilibrium potential. In neurons with a large membrane potential, the
electrical gradient for K+ was reduced during the membrane
hyperpolarization, preventing the enhancement of the amplitude of AH
(Fig. 1A). During the early phase of membrane
depolarization, an increased electrical gradient for K+ and
the remaining conductance of the Ca2+-dependent
K+ channel kept the amplitude of AH constant for a short
period before the peak of the membrane depolarization (Fig.
1B).
Reversal of HX/XO effects by scavengers.
To identify ROS responsible for the changes induced in the membrane
potential of AH/type 2 neurons, antioxidant scavengers such as SOD,
CAT, and DMSO were added to the superfusing solution with HX/XO. SOD
(38 U/ml), an enzyme that dismutates
O2·
to H2O2,
reversed neither the membrane hyperpolarization nor the depolarization
but augmented hyperpolarizing responses in five neurons (Fig.
2B). CAT (5 U/ml), an enzyme that decomposes H2O2, partially suppressed membrane
depolarization (Fig. 2C; n = 4). Application
of H2O2 (15 mM) by microejection from a
fine-tipped pipette positioned in the close vicinity of the impaled
neurons mimicked the effects of HX/XO. Microapplication of
H2O2 evoked a biphasic response in four
neurons, a transient membrane hyperpolarization in two neurons, and a
slow depolarization in three neurons (data not shown). On the other
hand DMSO (15 mM), a scavenger of OH·, suppressed both
hyperpolarizing and depolarizing responses by HX/XO (Fig.
8; n = 3). These results
indicated that OH· was the principal ROS involved in the
changes in the membrane excitability of AH/type 2 neurons produced by
HX/XO application. The effects of microapplication of
H2O2 could be explained by OH·
generated in Fenton reaction with H2O2 and
Fe2+ in the tissue (35).

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Fig. 8.
Effects of DMSO on HX/XO-induced membrane
hyperpolarization and depolarization. A: a transient
hyperpolarization (left) and a subsequent slow membrane
depolarization (right) were induced in an AH/type 2 neuron
by application of HX (10 µM) and XO (0.01 U/ml). B:
addition of DMSO (15 mM) in the superfusing Krebs solution before HX/XO
application significantly attenuated hyperpolarizing (left)
and depolarizing (right) responses. C: after the
tissue was washed with normal Krebs solution for 15 min,
hyperpolarizing response by HX/XO application recovered
(left), whereas depolarizing response was still suppressed
(right). Three steps of different hyperpolarizing currents
were injected at intervals of 10 s, and the fiber tract was
electrically stimulated every 30 s throughout the experiment.
Top traces, transmembrane potential; bottom
traces, injected current.
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Mimicry of HX/XO effects by caffeine.
Because HX/XO application changed the size of the AH that are dependent
on the cytosolic Ca2+ concentration and inhibited somatic
Ca2+ currents that form a part of action potentials, it was
speculated that intracellular Ca2+ homeostasis might be
altered by HX/XO-derived ROS. To test this possibility, caffeine, an
agonist for the Ca2+-dependent Ca2+-release
channels of the endoplasmic reticulum (ER), was applied to three
AH/type 2 neurons in the presence of 0.2 µM TTX in the superfusing
Krebs solution. Application of caffeine (2-10 mM) induced a
transient membrane hyperpolarization and a subsequent slow membrane
depolarization in a dose-dependent manner, mimicking the effects of
HX/XO application (Fig. 9). The average
membrane hyperpolarization induced by 10 mM caffeine was
13.3 ± 3.8 mV (n = 3). The changes in the input resistance
observed during caffeine application were identical to those induced by
HX/XO application (Fig. 9), suggesting the possibility that the
HX/XO-derived ROS or OH· might be acting on the
Ca2+ storage sites and thereby increasing the intracellular
concentration of Ca2+.

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Fig. 9.
Mimicry of HX/XO-induced effects by application of
caffeine. A: addition of caffeine (2 mM) in the superfusing
Krebs solution with the presence of TTX (0.2 µM) induced a transient
membrane hyperpolarization ( ) and a slow depolarization ( ) in an
AH/type 2 neuron (left). Changes in the membrane potential
to steps of hyperpolarizing currents shown on a fast time base indicate
a significant decrease ( ) and increase ( ) in the input resistance
compared with the control before HX/XO application (*)
(right). B and C: higher
concentrations of caffeine (B: 5 mM, C: 10 mM)
induced larger changes in the membrane potential and the input
resistance. Three steps of different hyperpolarizing currents were
injected at intervals of 8 s, and the fiber tract was electrically
stimulated every 32 s throughout the experiment. Left
panels: top traces, transmembrane potential;
bottom traces, injected current. Right panels:
membrane potential changes produced by 3 steps of hyperpolarizing
currents.
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DISCUSSION |
This study provides the first description of the effects of ROS on
AH/type 2 myenteric neurons in the guinea pig distal colon. The results
obtained clearly demonstrate that ROS derived from HX/XO reaction have
two direct actions on the postsynaptic membrane. The first response was
the transient membrane hyperpolarization that occurred during the early
phase of HX/HO application. The second response, slow membrane
depolarization, occurred during the late phase of HX/HO application
with an increase in the membrane excitability. This type of response
mimicked the slow excitatory postsynaptic potentials (EPSPs) that were
reported as the characteristic synaptic response in the enteric nervous
system (34). In addition, ROS suppressed Ca2+
current that forms a part of the inward currents of the somatic action
potentials without affecting the kinetics of voltage-dependent Na+ channels in the neurites. Complete suppression of
HX/XO-induced membrane hyperpolarization and depolarization by DMSO, a
scavenger of OH·, and mimicry by focal application of
H2O2 supported the assumption that
OH· is the responsible ROS for these responses.
AH/type 2 neurons.
The AH/type 2 neuron is one of the major electrophysiological subtypes
identified in the enteric nervous system. This type of neuron is
characterized by TTX-resistant action potentials and prolonged
afterhyperpolarizations (20, 27, 33, 34). Both
Na+ and Ca2+ channels carry the inward current
of the action potential of AH/type 2 neurons, whereas the outward
K+ current of AH is carried by Ca2+-dependent
K+ channels being activated by Ca2+ that has
entered during the action potential (20, 34).
Myenteric neurons with AH/type 2 electrophysiology have been identified
in the duodenum, ileum, proximal and distal colon, and rectum with some
differences in the distribution ratio to the total population of
neurons (27, 33, 34). No AH/type 2 neurons, however, were
found in the myenteric plexus of the stomach and gallbladder
(34). In the distal colon and rectum of the guinea pig,
AH/type 2 neurons account for ~24% of the total population of
myenteric neurons (27, 33). The intracellular staining
studies revealed that ~80% of these neurons have Dogiel type II
morphology, a large oval cell body with multiple long processes or
neurites (7, 16). The long processes of Dogiel type II
neurons in the small and large intestine form a number of varicose
terminals around other myenteric neurons, suggesting the
pivotal roles of this type of neurons in the enteric circuitry (4, 16). AH/type 2 neurons with Dogiel type II morphology in the small intestine have been implicated as primary sensory neurons
that respond to mechanical changes occurred in the mucosa (3). The functional role of AH/type 2 neurons in the
colonic enteric circuits, however, has not been well understood and
still remains to be investigated.
Ionic mechanism of ROS effects.
A patch-clamp study of the cultured AH/type 2 myenteric neurons from
the small intestine revealed the existence of four types of
K+ channels: Ca2+ dependent, A type, delayed
rectifying, and inwardly rectifying (36). Of these types,
the principal role of Ca2+-dependent K+
currents in determination of the membrane potential of myenteric AH/type 2 neurons has been repeatedly confirmed. Reduction of Ca2+ in the extracellular fluid or blockade of
Ca2+ entry by multivalent cations has been shown to
depolarize AH/type 2 neurons (34). A steady influx of
Ca2+ via low-voltage-activated Ca2+ (T type)
channels (24) is considered to be the main source of
intracellular free Ca2+ that keeps
Ca2+-dependent K+ channels open at the resting conditions.
Several putative neurotransmitters in the enteric nervous system have
been shown to modulate Ca2+-dependent K+
currents in the somal membrane of AH/type 2 neurons in the small and
large intestines (33, 34). Norepinephrine and galanin, a
29-amino acid gut peptide, induce a membrane hyperpolarization by
opening Ca2+-dependent K+ channels and mimic
slow inhibitory postsynaptic potentials (28, 34).
Acetylcholine, substance P, 5-hydroxytryptamine, histamine, and
calcitonin gene-related peptide reduce the conductance of Ca2+-dependent K+ channels and induce a slow
membrane depolarization (19, 34). The elevation of
intracellular levels of cAMP has been proposed as the intracellular
mechanism that leads to closure of Ca2+-dependent
K+ channels and initiation of slow membrane excitation
(21). The results obtained in this study clearly
demonstrated that the transient membrane hyperpolarization produced by
HX/XO-derived OH· is caused by the opening of
Ca2+-dependent K+ channels, whereas the slow
membrane depolarization is produced by closure of the same channels. An
interesting finding in this study was that SOD blocked neither the
membrane hyperpolarization nor the depolarization induced by HX/XO but
enhanced transient membrane hyperpolarization. Because SOD dismutates
O2·
to form
H2O2 (35), it is suggested that
the concentration of H2O2 and/or
OH· was increased by addition of SOD to HX/XO-containing
Krebs solution, enhancing the effects of OH· on the
membrane potential.
Several lines of evidence have suggested that ROS modulate
K+ channels but with an extremely complex pattern (8,
11). O2·
derived from HX/XO
reaction activates a subunit of G protein-activated inward rectifier
K+ channels expressed in oocytes (11). On the
other hand, the basal inward rectifier K+ channel, Shaker
K+ channels, and Shaw K+ channels were
inhibited by ROS generated by photoactivation of rose bengal but were
resistant to HX/XO application, indicating that different sources of
ROS have different effects on susceptible K+ channels
(8). The observations in this study, however, indicated that the inward rectifier K+ channels that form the
hyperpolarizing undershoot of the action potentials of myenteric
AH/type 2 neurons are resistant to HX/XO-derived ROS.
Mimicry of the ROS-induced responses by application of caffeine
supports the possibility that an initial increase of cytosolic Ca2+ concentration might be a trigger for the sequential
modulation of Ca2+-dependent K+ channels
produced by HX/XO application. The transient membrane hyperpolarization
by HX/XO-derived ROS can be explained by an increase of intracellular
Ca2+ concentration. However, the mechanism that leads to
closure of Ca2+-dependent K+ channels and
generation of slow depolarization appears to be complicated. There was
no correlation between HX concentration and the changes in the input
resistance measured during the membrane depolarization (Fig. 5).
Recovery of the HX/XO-induced slow membrane depolarization after
suppression by DMSO occurred more slowly than that of the
hyperpolarizing response (Fig. 8C). In murine vascular
smooth muscle cells, H2O2 has been shown to
enhance forskolin-stimulated adenylyl cyclase activity
(25). Therefore, it is speculated that ROS derived from
HX/XO reaction might directly enhance the production of cAMP or that
elevated intracellular Ca2+ might trigger secondary
processes that eventually decrease conductance of the
Ca2+-dependent K+ channels of the somatic membrane.
ROS generation.
The generation processes for ROS are tightly interacting and often
develop concurrently (6, 10, 15, 35). A number of
enzymatic and nonenzymatic sources produce ROS. In the gastrointestinal tract there are two possible enzymatic reactions that generate ROS. The
HX/XO reaction can be activated in epithelial and vascular endothelial
cells after ischemia-reperfusion of the tissue, and NADPH oxidase of
activated leukocytes in the site with inflammation produces
O2·
. In mammals, a high concentration
of xanthine dehydrogenase, an NAD-dependent form of XO, is localized in
the epithelial cells of the gastrointestinal tract (10).
During ischemia of the tissue, xanthine dehydrogenase is converted to
XO, an oxygen-dependent form, and ATP is catabolized to HX. During
reoxygenation of the tissue, XO degrades HX to uric acid and produces
O2·
.
O2·
is then converted to
H2O2 by one-electron oxidation and, in the presence of Fe2+, OH· is generated from
H2O2 by a Fenton reaction (15,
35). The increased production of ROS in the epithelium has been
suggested to change the balance of protective enzymes, inducing an
injury or ulceration in the mucosal layer (10). The
endothelial cells of the blood vessels in the gastrointestinal tract
also contain sufficient xanthine dehydrogenase to produce ROS. ROS
produced by HX/XO reaction in the endothelial cells have been
implicated in ischemia-reperfusion-induced microvascular dysfunction
and resultant mucosal injury in the small intestine (10).
Circulating neutrophils are recruited to sites of inflammation and
tissue injury by a highly regulated process. In the active regions of
IBD, a number of inflammatory cells infiltrate into the colonic
interstitium. A membrane-bound enzyme, NADPH oxidase, produces
O2·
for phagocytic activity, and >90%
of the oxygen consumed by activated neutrophils is secreted as
O2·
into the external environment
around the cells (9). Therefore, symptoms often observed
in patients with IBD, such as nausea, anorexia, diarrhea, and
constipation, might be the result of the motility disorder caused by
dysfunction of myenteric neuronal circuits that have been exposed to
ROS released from activated leukocytes.
ROS derived from HX/XO reaction stimulate mast cells to release
histamine (17). In the myenteric plexus of the guinea pig small intestine, histamine induces a slow membrane depolarization in
myenteric neurons by activating postsynaptic excitatory H1 and H2 receptors (19) and suppresses fast
EPSPs through activation of the presynaptic H3 receptor
(29). It is unlikely, however, that histamine released
from mast cells was involved in the generation of slow depolarization
by HX/XO-derived ROS. This is because the ED50 for
presynaptic inhibitory action of histamine is much lower than that for
histamine's postsynaptic excitatory effects (29), but in
our preliminary observation, no fast EPSPs evoked in the colonic
myenteric plexus were suppressed during application of HX/XO (data not shown).
ER as the target of ROS.
Although the exact origin of Ca2+ is still
controversial, ROS are known to increase cytosolic Ca2+ and
induce various biological processes through the Ca2+
signaling system (25). Macromolecules such as
Ca2+-releasing channels and ATP-dependent Ca2+
pumps of the sarcoplasmic reticulum are the possible target sites of
oxidant to increase cytosolic Ca2+ concentrations in
muscles. There are two types of Ca2+-releasing pathways
that are sensitive to exogenous ROS, ryanodine-sensitive Ca2+-release channels and inositol 1,4,5-trisphosphate
(IP3)-dependent Ca2+-release channels.
O2·
derived from HX/XO reaction
increases Ca2+ efflux from cardiac sarcoplasmic reticulum
by decreasing calmodulin, a calcium binding protein that inhibits
ryanodine-sensitive Ca2+ channels (14).
IP3-induced contraction in
-toxin-permeabilized vascular
smooth muscles is not affected by O2·
(32), but IP3-induced Ca2+ release
from sarcoplasmic reticulum of dissociated vascular muscles is
activated by O2·
(25). In
addition, the ATP-dependent Ca2+ pump, an enzyme that
transports cytosolic Ca2+ back into the intracellular store
sites against the concentration gradient, is inhibited by both
O2·
and H2O2
(25).
In myenteric AH/type 2 neurons, the amplitude and duration of AH
increase as the number of action potentials evoked by injection of a
depolarizing current pulse increases (20, 28). Application of tetraethylammonium, an inhibitor of delayed-rectifier K+
channels, broadens action potentials and enhances the amplitude and
duration of the AH, suggesting that Ca2+ influx via somal
Ca2+ channels (and a resultant increase of cytosolic
Ca2+) is the underlying mechanism for generation of AH
(28). The temperature dependence of the AH both in
activation and inactivation processes is indicative of the involvement
of a metabolic process rather than simple diffusion of Ca2+
(20, 34). Recent studies have shown that neurons contain intracellular stores of Ca2+ that can be released by
IP3, by caffeine, or by a rise in intracellular Ca2+ concentration (2, 25). In guinea pig
preganglionic motor neurons of the vagus, activation of
Ca2+-dependent K+ channels that underlies the
generation of AH is induced by Ca2+ released via
ryanodine-sensitive Ca2+ channels from the internal stores
(23). The delayed onset of AH suggests the existence of a
similar activation mechanism for Ca2+-dependent
K+ channels in myenteric AH/type 2 neurons. Membrane
hyperpolarization induced by caffeine in colonic AH/type 2 neurons is
compatible with the mechanism of Ca2+-induced
Ca2+ release from the neuronal ER and resultant activation
of Ca2+-dependent K+ channels.
In the rat heart muscle, ROS derived from HX/XO reaction have been
shown to directly reduce the number of Ca2+ channels in the
cell membrane and decrease the voltage-dependent Ca2+
influx (13). Our observations in the present study,
however, strongly suggest that the intracellular Ca2+ store
sites of the myenteric neurons are the plausible targets of ROS. An
initial efflux of Ca2+ by ROS from the intracellular stores
beneath the plasma membrane appears to trigger a transient membrane
hyperpolarization by opening Ca2+-dependent K+
channels. Further increase in the intracellular concentration of
Ca2+ might have induced a slow membrane depolarization by
activating cAMP production system or other intracellular mechanisms
that lead to closure of Ca2+-dependent K+
channels. Therefore, the reduction of the amplitude of Ca2+
spikes could be a result of a decreased concentration gradient for
Ca2+ across the somatic membrane. Factors such as the
amount of Ca2+ initially released by ROS and the state of
membrane excitability of the impaled neurons before HX/XO application
might have decided the pattern of response produced by ROS.
Physiological significance.
Our observations in this study add to earlier indications that
the actions of ROS on enteric neurons may be of important physiological and pathophysiological significance. The minimal concentration of HX
that could induce the changes in the membrane excitability of AH/type 2 neurons was <1 µM. This concentration of HX is much lower than those
used in earlier studies to induce Ca2+ release in
permeabilized smooth muscles (100 µM; Ref. 32) or from
the sarcoplasmic reticulum of heart muscles (20 µM; Ref. 14). Considering that the myenteric neurons are covered by
the basement membrane in our preparations, the actual concentration of
ROS that could reach the enteric neurons might be <1 µM and within
the range of physiological concentrations (12, 15). In
addition, all of the effects produced by HX/XO-derived ROS in AH/type 2 neurons were reversible. Therefore, our observations suggest that
OH· might be one of the neurotransmitters or
neuromodulators in the colonic enteric circuits. OH·
generated by O2·
released from
activated neutrophils, vascular endothelial cells, or epithelial cells
could modulate membrane excitability of myenteric neurons and thereby
alter the functional state of enteric neuronal circuits.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: K. Tamura, Dept. of Physiology, Kanagawa Dental College, 82 Inaoka-cho, Yokosuka 238-8580, Japan (E-mail:ktamura{at}kdcnet.ac.jp).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 13 December 1999; accepted in final form 3 May 2000.
 |
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