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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 MOmega ). 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Electrophysiological characteristics of colonic AH/type 2 neurons

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).


View larger version (29K):
[in this window]
[in a new window]
 
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.



View larger version (52K):
[in this window]
[in a new window]
 
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.


                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Effect of HX/XO on colonic AH/type 2 neurons



View larger version (26K):
[in this window]
[in a new window]
 
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).

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.


View larger version (22K):
[in this window]
[in a new window]
 
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 (open circle ) 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.

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.


View larger version (19K):
[in this window]
[in a new window]
 
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 (open circle ). Both values are expressed as a percentage of the control. Abscissa gives the concentration of HX (n = 3, mean ± SE).

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.


View larger version (11K):
[in this window]
[in a new window]
 
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.

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).


View larger version (10K):
[in this window]
[in a new window]
 
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).

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).


View larger version (25K):
[in this window]
[in a new window]
 
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.

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+.


View larger version (33K):
[in this window]
[in a new window]
 
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 (dagger ) and a slow depolarization (Dagger ) 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 (dagger ) and increase (Dagger ) 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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Babbs, CF, Cregor MD, Turek JJ, and Badylak SF. Endothelial superoxide production in the isolated rat heart during early reperfusion after ischemia. Am J Pathol 139: 1069-1080, 1991[Abstract].

2.   Berridge, MJ. Neuronal calcium signaling. Neuron 21: 13-26, 1998[ISI][Medline].

3.   Bornstein, JC, Furness JB, Smith TK, and Trussel DC. Synaptic responses evoked by mechanical stimulation of the mucosa in morphologically characterized myenteric neurons of the guinea-pig ileum. J Neurosci 11: 505-518, 1991[Abstract].

4.   Bornstein, JC, Hendriks R, Furness JB, and Trussell DC. Ramifications of the axons of AH-neurons injected with the intracellular marker biocytin in the myenteric plexus of the guinea pig small intestine. J Comp Neurol 314: 437-451, 1991[ISI][Medline].

5.   Cassarino, DS, and Bennett JP, Jr. An evaluation of the role of mitochondria in neurodegenerative diseases: mitochondrial mutations and oxidative pathology, protective nuclear responses, and cell death in neurodegeneration. Brain Res Rev 29: 1-25, 1999[ISI][Medline].

6.   Cross, AR, and Jones OTG Enzymic mechanisms of superoxide production. Biochim Biophys Acta 1057: 281-298, 1991[ISI][Medline].

7.   Dogiel, AS. Ueber den Bau der Ganglien in den Geflechten des Darmes und der Gallenblase des Menschen und der Säugethiere. Arch Anat Physiol Leipzig Anatomische Abteil 1899: 130-158, 1899.

8.   Duprat, F, Guillemare E, Romey G, Fink M, Lesage F, Lazdunski M, and Honore E. Susceptibility of cloned K+ channels to reactive oxygen species. Proc Natl Acad Sci USA 92: 11796-11800, 1995[Abstract].

9.   Fantone, JC, and Ward PA. Role of oxygen-derived free radicals and metabolites in leukocyte-dependent inflammatory reactions. Am J Pathol 107: 397-418, 1982[ISI].

10.   Granger, DN, Grisham MB, and Kvietys PR. Mechanisms of microvascular injury. In: Physiology of the Gastrointestinal Tract (3rd ed.), edited by Johnson LR.. New York: Raven, 1994, p. 1693.

11.   Jeglitsch, G, Ramos P, Encabo A, Tritthart HA, Esterbauer H, Groschner K, and Schreibmayer W. The cardiac acetylcholine-activated, inwardly rectifying K+ channel subunit GIRK1 gives rise to an inward current induced by free oxygen radicals. Free Radic Biol Med 26: 253-259, 1999[ISI][Medline].

12.   Jou, SB, and Cheng JT. The role of free radicals in the release of noradrenaline from myenteric nerve terminals of guinea-pig ileum. J Auton Nerv Syst 66: 126-130, 1997[ISI][Medline].

13.   Kaneko, M, Lee SL, Wolf CM, and Dhalla NS. Reduction of calcium channel antagonist binding sites by oxygen free radicals in rat heart. J Mol Cell Cardiol 21: 935-943, 1989[ISI][Medline].

14.   Kawakami, M, and Okabe E. Superoxide anion radical-triggered Ca2+ released from cardiac sarcoplasmic reticulum through ryanodine receptor Ca2+ channel. Mol Pharmacol 53: 497-503, 1998[Abstract/Free Full Text].

15.   Lacy, F, Gough DA, and Schmid-Schönbein GW. Role of xanthine oxidase in hydrogen peroxide production. Free Radic Biol Med 25: 720-727, 1998[ISI][Medline].

16.   Lomax, AEG, Sharkey KA, Bertrand PP, Low AM, Bornstein JC, and Furness JB. Correlation of morphology, electrophysiology and chemistry of neurons in the myenteric plexus of the guinea-pig distal colon. J Auton Nerv Syst 76: 45-61, 1999[ISI][Medline].

17.   Menon, IA, Shirwadkar S, and Ranadive NS. Nature of the oxygen species generated by xanthine oxidase involved in secretory histamine release from mast cells. Biochem Cell Biol 67: 397-403, 1988[ISI].

18.   Morrison, BM, and Morrison JH. Amyotrophic lateral sclerosis associated with mutations in superoxide dismutase: a putative mechanism of degeneration. Brain Res Rev 29: 121-135, 1999[ISI][Medline].

19.   Nemeth, PR, Ort CA, and Wood JD. Intracellular study of effects of histamine on electrical behaviour of myenteric neurones in guinea-pig small intestine. J Physiol (Lond) 355: 411-425, 1984[Abstract].

20.   North, RA, and Tokimasa T. Depression of calcium-dependent potassium conductance of guinea-pig myenteric neurones by muscarinic agonists. J Physiol (Lond) 342: 253-266, 1983[Abstract].

21.   Palmer, JM, Wood JD, and Zafirov DH. Elevation of adenosine 3',5'-phosphate mimics slow synaptic excitation in myenteric neurones of the guinea-pig. J Physiol (Lond) 376: 451-460, 1986[Abstract].

22.   Pellegrini-Giampietro, DE, Cherici G, Alesiani M, Carla V, and Moroni F. Excitatory amino acid release from rat hippocampal slices as a consequence of free-radical formation. J Neurochem 51: 1960-1963, 1988[ISI][Medline].

23.   Sah, P, and McLachlan EM. Ca2+-activated K+ currents underlying the afterhyperpolarization in guinea pig vagal neurons: a role for Ca2+-activated Ca2+ release. Neuron 7: 257-264, 1991[ISI][Medline].

24.   Starodub, AM, and Wood JD. Selectivity of omega -CgTx-MVIIC toxin from Conus magnus on calcium currents in enteric neurons. Life Sci 64: 305-310, 1999[ISI][Medline].

25.   Suzuki, YJ, Forman HJ, and Sevanian A. Oxidants as stimulators of signal transduction. Free Radic Biol Med 22: 269-285, 1997[ISI][Medline].

26.   Szabo, A, and Heemann U. Ischemia reperfusion injury and chronic allograft rejection. Transplant Proc 30: 4281-4284, 1998[ISI][Medline].

27.   Tamura, K. Morphology of electrophysiologically identified myenteric neurons in the guinea pig rectum. Am J Physiol Gastrointest Liver Physiol 262: G545-G552, 1992[Abstract/Free Full Text].

28.   Tamura, K, Palmer JM, Winkelmann CK, and Wood JD. Mechanism of action of galanin on myenteric neurons. J Neurophysiol 60: 966-979, 1988[Abstract/Free Full Text].

29.   Tamura, K, Palmer JM, and Wood JD. Presynaptic inhibition produced by histamine at nicotinic synapses in enteric ganglia. Neuroscience 25: 171-179, 1988[ISI][Medline].

30.   Tamura, K, and Wada-Takahashi S. Modulation of membrane excitability of colonic myenteric neuron by reactive oxygen species. In: Neurogastroenterology, edited by Singer MV, and Krammer H-J.. Dordrecht, The Netherlands: Kluwer Academic, 2000, p. 193 (Falk Symposium 112).

31.   Vliet, AVD, and Bast A. Role of reactive oxygen species in intestinal diseases. Free Radic Biol Med 12: 499-513, 1992[ISI][Medline].

32.   Wada, S, and Okabe E. Susceptibility of caffeine- and Ins (1,4,5)P3-induced contractions to oxidants in permeabilized vascular smooth muscle. Eur J Pharmacol 320: 51-59, 1997[ISI][Medline].

33.   Wade, PR, and Wood JD. Electrical behavior of myenteric neurons in the guinea pig distal colon. Am J Physiol Gastrointest Liver Physiol 254: G522-G530, 1988[Abstract/Free Full Text].

34.   Wood, JD. Physiology of the enteric nervous system. In: Physiology of the Gastrointestinal Tract (3rd ed.), edited by Johnson LR.. New York: Raven, 1994, p. 423.

35.   Yu, BP. Cellular defenses against damage from reactive oxygen species. Physiol Rev 74: 139-162, 1994[Free Full Text].

36.   Zholos, AV, Baidan LV, Starodub AM, and Wood JD. Potassium channels of myenteric neurons in guinea-pig small intestine. Neuroscience 89: 603-618, 1999[ISI][Medline].

37.   Zoccarato, F, Pandolfo M, Deana R, and Alexandre A. Inhibition by some phenolic antioxidants of Ca2+ uptake and neurotransmitter release from brain synaptosomes. Biochem Biophys Res Commun 146: 603-610, 1987[ISI][Medline].


Am J Physiol Gastrointest Liver Physiol 279(5):G893-G902
0193-1857/00 $5.00 Copyright © 2000 the American Physiological Society