Departments of 1 Pharmacology and Toxicology and 2 Medicine, and 3 Robarts Research Institute, University of Western Ontario, London, Ontario, Canada N6A 5C1
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ABSTRACT |
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Isolated myenteric ganglion networks were used in a perifusion protocol to characterize the response of interstitial adenosine levels to changes in prevailing PO2. The biological activity of such adenosine was assessed using inhibition of release of substance P (SP) as a functional measure of adenosine activity, and the effect of altered O2 tension on both spontaneous and elevated extracellular K+ concentration-evoked SP release from networks was determined over a range of PO2 values from hypoxic (PO2 = 54 mmHg) to hyperoxic (PO2 = 566 mmHg). Release of SP was found to be sensitive to PO2, and a linear graded relationship was obtained. Perifusion in the additional presence of the adenosine A1-receptor-selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) revealed considerable adenosinergic inhibition with an inverse exponential relationship and hyperoxic threshold PO2. Disinhibition of evoked SP release by DPCPX in the absence of TTX was double that observed in its presence, indicating a neural source for some of the adenosine released during hypoxia. A postulated neuroprotective role for adenosine is consistent with the demonstrated relationship between interstitial adenosine and prevailing O2 tension.
enteric nerves; hypoxia; neuroprotection
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INTRODUCTION |
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INTESTINAL ISCHEMIA contributes to many disorders of the gastrointestinal tract and has received increasing investigative attention lately. Substantial morbidity arises as a consequence of ischemia, and serious derangements of intestinal motor and secretory function, as well as more subtle effects, also arise (29). Diminished mesenteric blood flow as a consequence of atherosclerosis has long been recognized to contribute to intestinal disease, especially in the elderly, and ischemia-induced changes in enteric neural activity have been implicated in inflammatory bowel diseases, especially Crohn's disease (33). At one end of the spectrum of severity, a report of frank mesenteric infarction after cocaine self-administration has appeared (14), and intestinal ischemia is now recognized to be a serious potential complication of the abuse of this drug (30). At the other end, reports of disturbed motor function, including altered migrating myoelectric complex (MMC) cycling, as a result of ischemia (or ischemia and subsequent reperfusion), have appeared (12). Ischemia may be responsible for the motor and secretory sequelae of severe exercise, which are observed in marathon runners (13, 15), and may underlie several other disorders.
The involvement of the enteric nervous system (ENS) in ischemia-induced alterations of intestinal function has been addressed by several investigators. Both in vivo and in vitro studies have demonstrated that ischemic conditions result in altered intestinal motility in vivo (8, 10) or give rise to altered tissue responses in vitro (4) and that disturbances in enteric neural function may be involved. Yano et al. (37) have reported morphological changes to enteric nerves as a consequence of ischemia, although it is clear that functional deficits would almost certainly precede any derangements associated with such overt changes. In addition to acute and chronic disorders, the consequences of both ischemia and reperfusion are of considerable importance in small intestinal transplantation. Disrupted motor function after autotransplantation has been reported in animals, and temporal dissociation of MMC cycling in transplanted intestinal segments has been observed (24). Motor disruption after human small intestine transplantation has been reported anecdotally, and altered enteric neural function may be an important contributing factor (see Ref. 27 and citations therein).
Endogenous mechanisms that may serve to protect tissues from the consequences of ischemia (and reperfusion) have been described, although not as yet, in the ENS. Among such mechanisms, interstitial adenosine may serve a protective function, and both cardioprotective and neuroprotective roles for the nucleoside have been proposed (4, 25, 26, 32). Substantial evidence supporting a neuroprotective role for adenosine in the central nervous system (CNS) has accumulated, and several mechanisms by which its protective function at the CNS might arise have been suggested (7, 28). These include increasing blood flow, decreasing excitatory amino acid (EAA) release, hyperpolarization, decreased Ca2+ entry, and decreased free radical production. Moreover, activation of adenosine receptors by exogenous agonists has been shown to mimic the protective role of the nucleoside while adenosine receptor antagonists exacerbate ischemic damage (7). Enhancement of the binding of adenosine to A1 receptors using an allosterically active enhancer has also been shown to reduce ischemic damage in rat brain (11).
Mediation of the actions of adenosine by four receptor subtypes is firmly established, and their demonstrated or putative contribution to the range of effects of the nucleoside has been discussed (8, 32). Thus A1 receptors mediate hyperpolarization of central neurons, as well as the reduced Ca2+ influx, which yields diminished EAA release. Adenosine A2a receptors mediate vasodilation and decreased free radical release, whereas A2b receptors also mediate vasodilation (7). The A3 receptor has similarly been shown to mediate decreased free radical release and increased antioxidant activity (17).
The possibility that adenosine exerts a similar neuroprotective role in the ENS arises and has been suggested (20). Such a function for the nucleoside would require its presence in the interstitium of the enteric plexuses under ischemic or hypoxic conditions, and it is clear that increasing concentrations should be present during increasing ischemia or hypoxia. Such observations at the ENS are lacking. It was observed that hypoxia reduced [3H]ACh release from a guinea pig longitudinal muscle-myenteric plexus preparation (LMMP) and that the nonselective adenosine receptor antagonist theophylline partially reversed this reduction (19). We have recently demonstrated that interstitial adenosine is present, and exerts a tonic inhibitory tone, in a preparation of perifused guinea pig myenteric ganglion networks, as measured by the increased release of substance P [SP; SP-like immunoreactivity (SPLI)] in the presence of the selective adenosine A1-receptor-antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; see Ref. 20). It was consequently of considerable interest to determine whether the concentration of endogenous interstitial adenosine thus revealed varies with the prevailing O2 tension in the predicted inverse manner. It was therefore important to use this same approach to assess the characteristics of the relationship between O2 tension and endogenous adenosine levels in the ENS using a functional index of the adenosine concentration, and this was the primary objective of the present study. An additional objective was to obtain functional data supporting a neural source for the released adenosine.
Depolarization of enteric neural networks with elevated extracellular
K+ concentration
([K+]o)
undoubtedly evokes the release of several mediators such that the
responses determined by measurement of changes in the levels of any one
mediator represents the net result of several converging influences.
Although such measurement has allowed useful information about the
properties of enteric neural networks to be obtained and, by extension,
about the function of the intact ENS, determination of such changes in
the absence of an exogenous depolarizing influence offered the
possibility of obtaining a more "physiological" picture of
neuromediator release in myenteric networks. A parallel objective was
then to assess the feasibility of quantitating the spontaneous release
of SPLI from myenteric networks throughout all experiments and to
determine the relationship between unevoked SPLI release and prevailing
PO2.
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METHODS |
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Preparation of isolated myenteric neural networks. The procedure used to obtain isolated myenteric neural networks from the guinea pig ileum was as described by Moneta et al. (20). Briefly, the LMMP of one guinea pig (Hartley, 250-300 g, either sex; Charles River, Montreal, Quebec, Canada) was stripped and placed into Locke's solution of the following composition (in mM): 140 NaCl, 5 KCl, 5 NaHCO3, 1 MgCl2, 2.54 CaCl2, 10 dextrose, 10 HEPES, and 0.1% BSA, adjusted to pH 7.2 with NaOH. The LMMP strips were cut into 0.5-cm lengths and were placed in 30 ml Locke's solution with the following composition (in mg/100 ml): 167 collagenase (type IA), 133 protease (type IX from Bacillus polymyxa), 16 DNase I, and 30 bovine serum albumin (fraction V). The mixture was shaken in a warm water bath (37°C) for 30 min and then was centrifuged at 1,000 g for 10 min. The supernatant was removed, and the pellet was resuspended by gentle trituration with Locke's solution at 4°C. Ganglion networks were obtained by passing the solution through successive stainless steel mesh screens of 30, 50, and 150 mesh. The larger networks were trapped on the 50 mesh screen, and the smaller networks were trapped on the 150 mesh screen. Muscle tissue and other large debris were trapped by the 30 mesh screen and discarded. The networks were washed off the screens using Locke's solution, pooled, and loaded on 2.5-cm glass fiber filter disks (Whatman GF/F), placed in parallel perifusion chambers (Swinex filter holders; Millipore, Bedford, MA), and perifused with Locke's solution at 0.5 ml/min using a nonpulsatile roller pump (Gilson Minipuls 3; Mandel Scientific, Rockwood, ON, Canada). The temperature of the chambers, reservoirs, and tubing was maintained at 37°C by immersion in a large-volume water bath.
The perifusate collected during the first 28 min was discarded to allow
for equilibration. Subsequently, 1-ml samples of perifusate were
collected every 2 min in 1.5-ml Eppendorf tubes using a fraction collector, were heated to 98°C for 4 min, and were stored at
40°C until assayed for SPLI. In addition, total SPLI content
of the tissue on each filter was measured by heating the filter disk to
98°C in 1 ml of water for 4 min. The tube was then centrifuged at
13,500 g for 3 min, and the
supernatant was removed and stored at
40°C until assayed.
Experimental protocols. Evoked release
of SPLI was achieved by switching the perifusing solution from normal
Locke's solution to one containing an isotonic
[K+]o
(95 mM). Perifusing solutions, either normoxic (ungassed) or maintained
with altered PO2, contained all
necessary drugs appropriate for each experiment. All experiments were
performed using two chambers perifused in parallel, one serving as a
control for the other.
Altered PO2 was achieved using various O2/N2 gas mixtures. Locke's solutions were equilibrated by bubbling for 30 min before each experiment with the appropriate gas mixture, which was also maintained in the headspace above each reservoir throughout each experiment. "Hypoxic" conditions were created by bubbling with 100% N2, "hyperoxic" conditions were created with 100% O2, and intermediate O2 tensions were created with 10% O2, 20% O2, 30% O2, and 50% O2 (the remainder being N2). Solutions were equilibrated by bubbling with the gas(es) for 30 min using a glass wand fitted with a sintered glass disc and were then transferred to the appropriate reservoirs. The solutions were maintained at a constant PO2 using a slow bleed of the relevant gas mixture in the headspace throughout the experiment. Direct measurement of the solution PO2 was carried out after 1 h of storage in the reservoir, a time corresponding approximately to the middle of the experimental period. The conditions prevailing during experiments using ungassed Locke's solution were, for convenience, referred to as "normoxic," notwithstanding the acknowledged small discrepancy between the PO2 of such solutions and that of fully oxygenated blood.
RIA. The RIA procedure for SPLI has
been detailed by Brodin et al. (2). E. Brodin (Dept. of Pharmacology,
Karolinska Institute, Stockholm, Sweden) kindly supplied the SP-2
antibody, which detects 0.3 fmol/assay tube (10% drop from initial
binding) and cross-reacts with neurokinin A at 0.001% compared with SP
(100%). RIA standard curves were subjected to a log-logit transform
for analysis. The logit of the bound-to-free ratios (B/F),
(log{(B/F)/[K (B/F)]}), was then plotted against the log of the
concentration of standard peptide and a straight line fitted by least
squares linear regression (K is a
constant chosen to minimize deviation from linearity). Only standard
curve data points that deviated from the regression line by <10%
were accepted and used to interpolate the immunoreactivity present in
the samples. Standards were assayed in triplicate, whereas samples were
assayed in duplicate.
Data analysis. As described by Broad
et al. (1), evoked release of SPLI was determined by subtracting the
mean SPLI obtained in the five fractions immediately preceding the
measured period (i.e., mean unstimulated release) from the levels
obtained during the measured period (41-53 min). The cumulative
evoked release (CER) of SPLI was determined by summing the net release
and normalizing it to the total SPLI content on the filter disks. This
permitted an integral of the area under the release profile for all
points above that of the mean basal release to be determined.
Spontaneous SPLI release was calculated as the cumulative basal release
(CBR), which represented the integral of SPLI release obtained during the unstimulated period (29-39 min) before addition of
[K+]o,
normalized to the total SPLI content. Algebraically, the CBR was
calculated as
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Materials. With the following exceptions, all chemicals and reagents were obtained from BDH (Toronto, ON). HEPES, collagenase (type I), protease (type IX), DNase I, and TTX were obtained from Sigma Chemical (St. Louis, MO). DPCPX was obtained from Research Biochemicals (Natick, MA). 125I-labeled SP was purchased from DuPont (Markham, ON). All other reagents used for RIA were detailed by Brodin et al. (2). Solution PO2 measurements were obtained using an I-Stat (EG7+) Portable Clinical Analyzer (Hewlett Packard, Princeton, NJ).
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RESULTS |
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Release of SPLI from perifused myenteric ganglion
networks under hypoxic and hyperoxic conditions in the absence and
presence of DPCPX. Perifusion of isolated enteric
ganglion networks with Locke's solution equilibrated with 100%
N2 yielded a significant reduction
in both spontaneous and K+-evoked
SPLI release compared with control release. The CER and CBR values are
given in Table 1, and the corresponding
perifusion profiles are shown in Fig.
1A.
Perifusion of the networks with Locke's solution equilibrated with
100% N2 in the additional
presence of DPCPX (5 µM) resulted in a significant increase in both
spontaneous and K+-evoked SPLI
release compared with that in the absence of the adenosine
A1 receptor antagonist. The CER
and CBR values are shown in Table 1, and the corresponding perifusion
profiles are shown in Fig. 1B.
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Perifusion of networks in Locke's solution equilibrated with 100% O2 yielded a significant increase in K+-evoked SPLI release, whereas spontaneous release was not significantly altered. Perifusion in Locke's solution equilibrated with 100% O2 in the additional presence of DPCPX (5 µM) did not alter either the spontaneous or K+-evoked release of SPLI. The CER and CBR values are given in Table 1, and the corresponding perifusion profiles are shown in Fig. 1, C and D.
Release of SPLI from perifused myenteric ganglion networks under
conditions of increasing PO2 in
the absence and presence of DPCPX.
Perifusion of networks in Locke's solutions equilibrated with 10, 20, 30, or 50% O2 yielded the
spontaneous and K+-evoked SPLI
release shown by the appropriate CBR and CER values in Table 1. The
perifusion profiles corresponding to 10, 20, 30, and 50%
O2 are shown in Fig.
2, A,
C,
E, and
G, respectively. The CBR and CER
values obtained for perifusion under these conditions in the additional
presence of DPCPX (5 µM) are also shown in Table 1. The perifusion
profiles corresponding to all of these conditions in the additional
presence of DPCPX are shown in Fig. 2,
B, D, F, and
H, respectively.
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DISCUSSION |
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The primary objective of this study was to characterize the relationship between prevailing O2 tension and a functional measure of the concentration of endogenous adenosine present in the interstitium of myenteric neural networks. We report here evidence supporting the existence of an inverse graded relationship between endogenous adenosine concentration and O2 tension, as well as for the existence of a threshold O2 tension above which adenosine does not exert significant effects. Evidence supporting a tonic adenosine-mediated inhibitory tone within the networks is presented together with support for a neural source for some of the released nucleoside. A parallel objective was to examine the spontaneous (i.e., unevoked) release of excitatory mediator as a possibly more relevant variable with which to measure functionally the inhibition exerted by endogenous adenosine. The findings, which suggest that spontaneous release may indeed be a more sensitive and accurate measure of the actions of endogenous adenosine, are similarly reported here.
Spontaneous and evoked release of SPLI from isolated perifused
myenteric neural networks under normoxic conditions was not different
from that recently reported (20). In these previous studies, we had
quantified evoked release from networks as the integral CER, but had
not formally addressed the spontaneous release of SPLI. In the present
study, the integral of the release above baseline occurring
spontaneously during the 10 min preceding addition of the depolarizing
stimulus was calculated for all experiments as the CBR. The results
show that, not only could low levels of spontaneous SPLI release be
measured using the RIA technique but such release could be quantified
consistently using this approach. The CBR values reported here
demonstrate that changes in spontaneous release after experimental
intervention usually paralleled similar changes in evoked release and,
moreover, often yielded relative changes of greater magnitude. Because
the mediators released from myenteric networks include several distinct
entities, and the release of SPLI measured here is undoubtedly the net
result of several convergent inhibitory and excitatory influences,
neither CER nor CBR represents an exact model of intact enteric neural activity. However, the integral of spontaneous release is perhaps a
better model than that provided by analysis of the
[K+]o-evoked
release from isolated myenteric networks.
We had previously shown enhancement of release of SPLI by DPCPX, which constituted evidence for the presence of an endogenous inhibitory tone exerted, in part, by adenosine (20). We had also provided evidence that the primary contributor to that tone was indeed adenosine after enhancement of its actions by inhibition of nucleoside transport and reversal after incubation with the metabolizing enzyme adenosine deaminase. In the present experiments, perifusion under hypoxic conditions (100% N2) yielded reductions in both spontaneous and evoked release (Fig. 1A). In contrast, perifusion under hyperoxic conditions (100% O2) yielded increases in both spontaneous and evoked SPLI release (Fig. 1C). In the additional presence of the A1 antagonist DPCPX, the release of SPLI increased three- and fivefold for CER and CBR, respectively (Fig. 1B), revealing a substantial contribution of interstitial adenosine acting at A1 receptors to the overall inhibitory tone. Similar experiments conducted under hyperoxic conditions yielded no change in either spontaneous or evoked release (Fig. 1D), indicating that functionally undetectable levels of interstitial adenosine were present. The sensitivity of the release of adenosine from its source(s) to prevailing O2 tension is clearly demonstrated by this finding.
Perifusion of myenteric networks with solutions prepared at intermediate O2 tensions (10-50% O2) yielded responses that were intermediate to those obtained with the hypoxic and hyperoxic extremes. Inhibition was seen at 10% O2, little change was observed at both 20 and 30% O2, and enhancement of release was seen at 50% O2 (Fig. 2 and Table 1). In the additional presence of DPCPX, clear increments in both spontaneous and evoked release of SPLI occurred at both 10 and 20% O2, whereas, at 30 and 50% O2, the antagonist did not alter the release, and both the CER and CBR values remained essentially the same as those obtained with 100% O2. Examination of the association between PO2 and release of SPLI from myenteric networks revealed the clear graded relationships shown in Fig. 3, A and B. When measured PO2 values were plotted against the CER and CBR values expressed as percentages of their respective normoxic controls, the relationship obtained was amenable to linear regression analysis yielding reasonable linearity and correlations for both. However, such mathematical linearity does not necessarily imply biological linearity, and it is clear that the observed relationship arose as the net result of converging inhibitory and excitatory influences (20). It is tempting to speculate that the higher slope exhibited by the relationship for evoked release (CER) may reflect an increased concentration of PO2-sensitive inhibitory mediators in the interstitium, such as nitric oxide, vasoactive intestinal peptide, pituitary adenylate cyclase-activating peptide, and/or galanin (21, 34), when compared with those present under resting conditions. This contention is consistent with the substantially larger release of SPLI seen after depolarization. Notwithstanding this possibility, the contribution of adenosine to the overall PO2-sensitive inhibitory tone could not be addressed by examination of this relationship, and we therefore assessed the change in release of SPLI that occurred in the presence of adenosine receptor blockade.
When expressed as a percentage of control release in the absence of the A1 antagonist, the release in its presence at each PO2 revealed the magnitude of inhibition that had been due to the presence of interstitial adenosine alone. Figure 4, A and B, shows the exponential increases in the incremented release obtained with decreasing PO2 after the attainment of a threshold O2 tension. The increment in spontaneous release at low PO2 values (Fig. 4B) is greater than that observed for evoked release (Fig. 4A), suggesting that endogenous adenosine contributes a greater proportion of the overall inhibitory tone present in the networks under resting conditions than it does to that which exists after general depolarization.
A threshold for the onset of measurable adenosine-dependent inhibition was obtained for both spontaneous and evoked SPLI release. It is evident that, as PO2 falls from somewhere between 260 and 146 mmHg (i.e., between 30 and 20% O2), the influence of increasing interstitial adenosine becomes apparent for both spontaneous and evoked SPLI release. It may be useful to consider the biological relevance of the existence of this threshold, as well as the specific characteristics of the release. The existence of a threshold, and its occurrence at a relatively hyperoxic PO2, is consistent with both putative physiological and pathophysiological roles for the nucleoside in the tonic regulation of neuromediator release. It is particularly noteworthy that detectable endogenous adenosine levels are clearly present under both normoxic conditions (i.e., ungassed) and after perifusion with solution equilibrated at 20% O2 (Figs. 2D and 4). These findings indicate that adenosine contributes to the inhibitory tone in myenteric networks at PO2 values corresponding to those that presumably would occur were perfusion of isolated networks with fully oxygenated blood possible. By extension, the prevailing PO2 within the ENS in vivo, a parameter that seems to have escaped definitive scrutiny, would not be expected to be very different and may indeed be considerably lower, both normally and certainly under those pathophysiological circumstances alluded to above.
The concentration of endogenous adenosine present in the vicinity of
its receptors on enteric nerves is not known. However, estimates for
interstitial adenosine levels within the myocardium and the CNS have
been reported using several approaches (4, 23, 31). The values
determined for interstitial adenosine in the CNS are reported to be in
the vicinity of 10-300 nM, which are consistent with (unpublished)
observations from this laboratory showing an
EC50 for adenosine on the isolated
LMMP preparation, in the presence of nucleoside transport inhibition,
of 5.2 × 108 M. Pharmacologically, for adenosine to provide biologically useful regulation of mediator release in the ENS, its concentration in the
vicinity of its receptors should ideally coincide with the steep
portion of the sigmoid dose-response curve, permitting small changes in
concentration to elicit the greatest change in response. The results
presented here are consistent with this possibility.
The source of the adenosine released in the interstitium of myenteric networks was addressed in an additional series of experiments. The myenteric network preparation is acknowledged to be a mixed tissue in which smooth muscle and other contaminating cells are present together with enteric neural tissue, itself a mixed tissue consisting of neurons, glia, and other support cells. All of these cells are potential sources of released adenosine, although the preponderant tissue appears to be derived from the ENS (20). We therefore used TTX to assess functionally the contribution of conducted nerve traffic to the release of endogenous adenosine.
Perifusion of networks in the presence of 1 µM TTX yielded a
substantial reduction in spontaneous release with little effect on
evoked release (Figs. 5A and
6B and Table 2). It should be pointed
out that this result was at variance with our previous finding showing
an increase in CER in the presence of the sodium channel blocker. We
were initially surprised at this discrepancy until repetition of the
experiment with a lower concentration of TTX revealed that the
concentration reported by Moneta et al. (20) as 1.0 µM was, in fact,
0.1 µM. Use of this lower concentration in the present experiments
reproduced the previously reported enhanced release exactly. It is
beyond the scope of this discussion to examine explanations for the
clear difference in response to TTX other than to suggest that
differential sensitivity of smaller-diameter inhibitory nerve fibers,
bearing lower densities of sodium channels, could be implicated. This
interesting possibility might provide a useful approach to the
functional investigation of distinct populations of enteric nerves but
will not be pursued here. Notwithstanding these considerations, the
substantial reduction in SPLI release brought about by 1.0 µM TTX was
observed in four separate experiments and was not affected by
perifusion under hypoxic (100%
N2) conditions (Figs.
5B and
6B and Table 2). We had already shown
that the inhibited spontaneous and evoked SPLI release under hypoxic
conditions could be essentially restored by perifusion with DPCPX
(Figs. 1B and
6C), and repetition of this
experiment in the additional presence of TTX was undertaken. The
results showed that, in the presence of TTX, the ability of DPCPX to
restore the inhibited evoked release of SPLI was diminished, yielding a
1.5-fold increment in CER over its control value (Table 2). By
contrast, in the absence of TTX, DPCPX incremented evoked release
approximately threefold (Figs. 1B and
6C and Table 1). Although these values were both different from their corresponding controls, the biological significance of the differences is perhaps more important than the
statistical differences. The
[K+]o-evoked
release of SPLI from myenteric networks occurs by depolarization not
only of nerve cell bodies but also directly at the nerve varicosities, a process that has long been recognized to be insensitive to TTX. Thus
the small decrement in CER with TTX and the similarly small increment
in the additional presence of DPCPX are expected findings. Because
DPCPX was less able to disinhibit evoked release in the presence of TTX
than in its absence, the interstitial adenosine that exerted the
inhibition in the latter case must have been released by a process
involving conducted action potentials, and thus the source of such
adenosine was neural. The observation that there remains an increment
in evoked release in the presence of both TTX and DPCPX indicates that
some interstitial adenosine must have been inhibiting release, and thus
only a portion of the overall interstitial adenosine originated from
those nerves sensitive to TTX, at least under the conditions of these experiments.
The precise identity of the myenteric neurons expressing A1 receptors and releasing SP as mediator was not addressed in this study. The presence of A1 receptors on AH/type 2 enteric neurons has been firmly established (3), whereas SP immunoreactivity has been localized in sensory neurons exhibiting type II morphology (6). Because type II myenteric neurons constitute ~30% of guinea pig ileal myenteric neurons, it is likely that they contributed to the SPLI release measured in the present experiments. However, other classes of myenteric neurons display SP immunoreactivity, and their contribution to the overall SPLI release measured here may also be relevant.
Although neural release of adenosine per se is implied from these studies, an additional mechanism is likely to be involved. We have previously shown that metabolism of ATP by ecto-ATPases yields adenosine, which contributes to the overall endogenous inhibitory tone (20), and the neural origin of the majority of such ATP is supported by its role as a fast transmitter in the ENS (9). Moreover, in addition to the known release of ATP from enteric synaptosomes (35), McConalogue et al. (18) have recently reported direct measurement of the release of the nucleotide from nerves in guinea pig taenia coli.
The findings reported here reflect changes in the release of mediator from myenteric networks under hypoxic but not strictly ischemic conditions. It is recognized that the consequences of diminished mesenteric blood flow and/or increased intestinal metabolic demand are not necessarily modeled by simple alteration of PO2. Nevertheless, tissue hypoxia is an important component of the changes brought about by ischemia in the environment of the ENS, and the present findings show that, under such conditions, progressively diminishing tissue PO2 will elicit exponentially increasing concentrations of interstitial adenosine. The presence of such adenosine in the vicinity of its receptors, and in functionally relevant concentrations, is compatible with a putative neuroprotective role for adenosine, the retaliatory metabolite (22). It is in the right place, potentially at the right time, and in appropriate amounts.
The role of adenosine as a putative neuroprotective moiety in the ENS necessarily follows from the emerging evidence for such a function in the CNS (25, 26, 32). In particular, the ability of the nucleoside to provide protection against acute EAA toxicity is worthy of consideration. Glutamatergic transmission within the ENS is recognized (36), and the excitotoxic effects of glutamate on both isolated enteric ganglia and enteric whole mount preparations have been reported (16). Interestingly, increased excitation after acute ischemia/hypoxia has been documented (5, 10), and, although such excitation may not involve glutamate exclusively, the parallels between such acute responses at the CNS and those emerging at the ENS suggest that exploration of the possible protective role of endogenous adenosine at this locus may be useful.
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ACKNOWLEDGEMENTS |
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We thank Dr. S. Mathur, Dept. of Anesthesiology, London Health Sciences Center, for kind assistance with measurement of solution PO2.
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FOOTNOTES |
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These studies were supported by operating grants from the Medical Research Council of Canada to M. A. Cook and T. J. McDonald. N. A. Deshpande is a member of the University of Western Ontario Graduate Program in Neuroscience.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. A. Cook, Dept. of Pharmacology & Toxicology, Univ. of Western Ontario, London, Ontario, Canada, N6A 5C1 (E-mail: mcook{at}julian.uwo.ca).
Received 15 September 1998; accepted in final form 5 December 1998.
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