Endogenous interstitial adenosine in isolated myenteric neural networks varies inversely with prevailing PO2

N. A. Deshpande1, T. J. McDonald1,2,3, and M. A. Cook1

Departments of 1 Pharmacology and Toxicology and 2 Medicine, and 3 Robarts Research Institute, University of Western Ontario, London, Ontario, Canada N6A 5C1


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 (up-arrow [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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 up-arrow [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 up-arrow [K+]o, normalized to the total SPLI content. Algebraically, the CBR was calculated as
CBR = <LIM><OP>∑</OP><LL><IT>t = i</IT></LL><UL><IT>j</IT></UL></LIM> <FR><NU>R<SUB><IT>t</IT></SUB></NU><DE>C<SUB>f</SUB></DE></FR> × 100/10 (%/min)
where Rt is SPLI released at time t, i is 29 min, j is 39 min, and Cf is total SPLI content on the filter. As previously reported (20), CER was calculated as
CER = <LIM><OP>∑</OP><LL><IT>t = i</IT></LL><UL><IT>j</IT></UL></LIM> <FR><NU>(R<SUB><IT>t</IT></SUB> − <OVL>&ugr;</OVL>)</NU><DE>C<SUB>f</SUB></DE></FR> × 100/14 (%/min)
where i is 41 min, j is 53 min, and <OVL>&ugr;</OVL> is mean unstimulated release. Each experiment was carried out using tissue from separate animals, and data from each experiment were pooled with equivalent data from at least three separate experiments. Statistical analysis of SPLI release under control and experimental conditions was performed using a two-tailed Student's t-test for single parameters (Graph Pad Instat, GraphPad Software, San Diego, CA, and Microsoft Excel 97, Microsoft, Seattle, WA). Paired Student's t-tests were also performed as appropriate. Statistical significance was accepted at P < 0.05.

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).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Table 1.   Release of SPLI from perifused networks of myenteric ganglia at various oxygen tensions with and without the additional presence of DPCPX



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Fig. 1.   Perifusion profiles showing release of substance P-like immunoreactivity (SPLI) from perifused myenteric ganglion networks. Spontaneous release was measured from 29 to 39 min (horizontal dotted line), and evoked release was measured from 41 to 53 min (horizontal dashed line) in this and Figs. 2-6. Elevated extracellular K+ concentration (up-arrow [K+]o) was present from 42 to 52 min (horizontal solid line). A: perifusion profiles showing SPLI release under normoxic conditions and after equilibration with 100% N2 (hypoxia; n = 5 separate experiments). B: perifusion profiles showing SPLI release under hypoxic conditions and in additional presence of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 5 µM; n = 3 separate experiments). C: perifusion profiles showing SPLI release under normoxic conditions and after equilibration with 100% O2 (hyperoxia; n = 3 separate experiments). D: perifusion profiles showing release of SPLI under hyperoxic conditions and in the additional presence of DPCPX (5 µM; n = 3 separate experiments). For Figs. 1-6, bars are means ± SE. Where no bars are shown, value was less than the datum point dimension.

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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Fig. 2.   A, C, E, and G: perifusion profiles showing SPLI release from perifused myenteric networks under normoxic and relatively hypoxic or hyperoxic conditions. Profiles shown are after equilibration with 10, 20, 30, and 50% O2, respectively, compared with normoxic controls. B, D, F, and H: perifusion profiles showing SPLI release under normoxic and relatively hypoxic/hyperoxic conditions and in the additional presence of DPCPX (5 µM; n = 3 separate experiments for all panels except A and E where n = 4).

Comparison of spontaneous and K+-evoked SPLI release values for each condition within the range of O2 tensions and expressed as a percentage of a normoxic (ungassed) control is shown in Fig. 3, A and B. The histogram includes a linear regression analysis with 95% confidence intervals. Measured PO2 values for Locke's solutions equilibrated with 100% N2 and 10, 20, 30, 50, and 100% O2 and for ungassed solution are also shown in Fig. 3, inset.


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Fig. 3.   A: histogram showing cumulative evoked release (CER) values for evoked SPLI release from perifused myenteric networks under conditions of increasing O2 tension. Release is expressed as percentage of control release under normoxic conditions (defined as 100%). Relationship was subjected to linear regression analysis, and the best-fit line with 95% confidence limits is shown. Best-fit line is y = 0.35x + 29 and r2 = 0.88. B: histogram showing equivalent cumulative basal release (CBR). Best-fit line is y = 0.22x + 27 and r2 = 0.94. Both slopes are significantly different from 0 (P < 0.05). Inset: measured PO2 values for all perifusing solutions equilibrated with the indicated gas mixtures and for ungassed (normoxic) solution.

Comparison of the release values for spontaneous and up-arrow [K+]o-evoked SPLI release in the presence of DPCPX expressed as a percentage of control values, for each condition within the range of O2 tensions used, is shown in Fig. 4, A and B. The scale of the abscissa has been reversed to present an increasing degree of hypoxia along the usual sense of the axis. A threshold for the increment in release is clearly present for networks perifused with Locke's solution equilibrated with between 30 and 20% O2, corresponding to PO2 values of between 260 and 146 mmHg.


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Fig. 4.   A: histogram showing CER in the presence of DPCPX (5 µM, CERDPCPX) under conditions of decreasing O2 tension (note reverse sense of abscissa). Release is expressed as percentage of control release in the absence of the antagonist (defined as 100%, horizontal dotted line). B: histogram showing CBR in the presence of DPCPX (5 µM, CBRDPCPX). * Significantly different from control (P < 0.05).

Release of SPLI from perifused myenteric ganglion networks under normoxic and hypoxic conditions in the absence and presence of TTX and, under hypoxic conditions, with or without the additional presence of DPCPX. Perifusion of networks with either normoxic Locke's solution or with Locke's solution equilibrated with 100% N2 in the absence or presence of TTX (1 µM) showed that TTX yielded a significant reduction in spontaneous SPLI release, whereas K+-evoked SPLI release was not altered significantly. The CER and CBR values are given in Table 2, and the corresponding perifusion profiles are shown in Fig. 5, A and B. Perifusion with Locke's solution equilibrated with 100% N2 and containing TTX (1 µM) in the absence or presence of DPCPX (5 µM) showed that the antagonist produced significant increases in the CER and CBR. The values are given in Table 2, and the corresponding perifusion profiles are shown in Fig. 5C. Graphical comparisons of these data are shown in Fig. 6. The CBR and CER values for spontaneous and K+-evoked SPLI release under normoxic and hypoxic conditions in the absence and presence of TTX are shown in Fig. 6, A and B. For the hypoxic condition, the CER values in the absence and presence of TTX (1 µM) and/or DPCPX (5 µM) are shown in Fig. 6C.

                              
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Table 2.   Release of SPLI from perifused networks of myenteric ganglia under normoxic and hypoxic conditions, in the presence of TTX, and in the additional presence of DPCPX



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Fig. 5.   A: perifusion profiles showing release of SPLI from perifused myenteric networks under normoxic conditions alone and in the additional presence of TTX (1 µM; n = 4 separate experiments). B: perifusion profiles showing release of SPLI under hypoxic conditions alone and in the additional presence of TTX (1 µM; n = 3 separate experiments). C: perifusion profiles showing release of SPLI under hypoxic conditions in the presence of TTX (1 µM) alone and in the additional presence of DPCPX (5 µM; n = 3 separate experiments).


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Fig. 6.   A: bar graph showing CER for SPLI release under normoxic (left) and hypoxic (right) conditions in the absence and presence of TTX (1 µM; n = 4 separate experiments). B: bar graph showing CBR for SPLI release under normoxic (left) and hypoxic (right) conditions in the absence and presence of TTX (1 µM; n = 3 separate experiments). C: bar graph showing CER for SPLI release under hypoxic conditions in the absence (left) and presence (right) of TTX (1 µM) and, for each, in the additional absence or presence of DPCPX (5 µM; n = 3 separate experiments). * Significant difference from paired control (P < 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 up-arrow [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 × 10-8 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 up-arrow [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.


    ACKNOWLEDGEMENTS

We thank Dr. S. Mathur, Dept. of Anesthesiology, London Health Sciences Center, for kind assistance with measurement of solution PO2.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Broad, R. M., T. J. McDonald, and M. A. Cook. Adenosine and 5-HT inhibit substance P release from nerve endings in myenteric ganglia by distinct mechanisms. Am. J. Physiol. 264 (Gastrointest. Liver Physiol. 27): G454-G461, 1993[Abstract/Free Full Text].

2.   Brodin, E., N. Lindefors, C. J. Dalsgaard, E. Theodorsson-Norheim, and S. Rosell. Tachykinin multiplicity in rat central nervous system as studied using antisera raised against substance P and neurokinin A. Regul. Pept. 13: 253-272, 1986[Medline].

3.   Christofi, F. L., and J. D. Wood. Electrophysiological subtypes of inhibitory P1 purinoceptors on myenteric neurones of guinea-pig small bowel. Br. J. Pharmacol. 113: 703-710, 1994[Abstract].

4.   Cook, M. A., and M. Karmazyn. Cardioprotective actions of adenosine and adenosine analogs. In: Myocardial Ischemia: Mechanisms, Reperfusion, Protection, edited by M. Karmazyn. Basel, Switzerland: Birkhauser, 1996, p. 325-344.

5.   Corbett, A. D., and G. M. Lees. Depressant effects of hypoxia and hypoglycaemia on neuro-effector transmission of guinea-pig intestine studied in vitro with a pharmacological model. Br. J. Pharmacol. 120: 107-115, 1997[Abstract].

6.   Costa, M., S. J. Brookes, P. A. Steele, I. Gibbins, E. Burcher, and C. J. Kandiah. Neurochemical classification of myenteric neurons in the guinea-pig ileum. Neuroscience 75: 949-967, 1996[Medline].

7.   Fredholm, B. B. Adenosine, and neuroprotection. Int. Rev. Neurobiol. 40: 259-280, 1997[Medline].

8.   Fredholm, B. B., M. P. Abbracchio, G. Burnstock, J. W. Daly, T. K. Harden, K. A. Jacobson, P. Leff, and M. Williams. Nomenclature and classification of purinoceptors. Pharmacol. Rev. 46: 143-156, 1994[Medline].

9.   Galligan, J. J., and P. P. Bertrand. ATP mediates fast synaptic potentials in enteric neurons. J. Neurosci. 14: 7563-7571, 1994[Abstract].

10.   Guisan, Y. J., A. Hreno, and F. N. Gurd. Effect of acute ischemia on the motility of the small bowel in the awake dog. Eur. Surg. Res. 7: 23-33, 1975[Medline].

11.   Halle, J. N., C. E. Kasper, J. M. Gidday, and B. J. Koos. Enhancing adenosine A1 receptor binding reduces hypoxic-ischemic brain injury in newborn rats. Brain Res. 759: 309-312, 1997[Medline].

12.   Hebra, A., M. F. Brown, K. McGeehin, D. Broussard, and A. J. Ross. The effects of ischemia and reperfusion on intestinal motility. J. Pediatr. Surg. 28: 362-365, 1993[Medline].

13.   Heer, M., F. Repond, A. Hany, H. Sulser, O. Kehl, and K. Jager. Acute ischaemic colitis in a female long distance runner. Gut 28: 896-899, 1987[Abstract].

14.   Hon, D. C., L. J. Salloum, H. W. Hardy, and J. E. Barone. Crack-induced enteric ischemia. N. Engl. J. Med. 87: 1001-1002, 1990.

15.   Kam, L. W., W. E. Pease, and P. D. Thompson. Exercise-related mesenteric infarction. Am. J. Gastroenterol. 89: 1899-1900, 1994[Medline].

16.   Kirchgessner, A. L., M. T. Liu, and F. Alcantara. Excitotoxicity in the enteric nervous system. J. Neurosci. 17: 8804-8816, 1997[Abstract/Free Full Text].

17.   Maggirwar, S. B., D. N. Dhanraj, S. M. Somani, and V. Ramkumar. Adenosine acts as an endogenous activator of the cellular antioxidant defense system. Biochem. Biophys. Res. Commun. 201: 508-515, 1994[Medline].

18.   McConalogue, K., L. Todorov, J. B. Furness, and D. P. Westfall. Direct measurement of the release of ATP and its major metabolites from the nerve fibres of the guinea-pig taenia coli. Clin. Exp. Pharmacol. Physiol. 23: 807-812, 1996[Medline].

19.   Milusheva, E., B. Sperlagh, B. Kiss, L. Szporny, E. Pasztor, M. Papasova, and E. S. Vizi. Inhibitory effect of hypoxic condition on acetylcholine release is partly due to the effect of adenosine released from the tissue. Brain Res. Bull. 24: 369-373, 1990[Medline].

20.   Moneta, N. A., T. J. McDonald, and M. A. Cook. Endogenous adenosine inhibits evoked substance P release from perifused networks of myenteric ganglia. Am. J. Physiol. 272 (Gastrointest. Liver Physiol. 35): G38-G45, 1997[Abstract/Free Full Text].

21.   Murthy, K. S., J. R. Grider, J. G. Jin, and G. M. Makhlouf. Interplay of VIP and nitric oxide in the regulation of neuromuscular function in the gut. Ann. NY Acad. Sci. 805: 355-362, 1996[Medline].

22.   Newby, A. C., Y. Worku, and C. A. Holmquist. Adenosine formation. Evidence for a direct biochemical link with energy metabolism. Adv. Myocardiol. 6: 273-284, 1985[Medline].

23.   Phillis, J. W., G. A. Walter, and R. E. Simpson. Brain adenosine and transmitter amino acid release from the ischemic rat cerebral cortex: effects of the adenosine deaminase inhibitor deoxycoformycin. J. Neurochem. 56: 644-650, 1991[Medline].

24.   Quigley, E. M., A. D. Spanta, S. G. Rose, J. Lof, and J. S. Thompson. Long-term effects of jejunoileal autotransplantation on myoelectrical activity in canine small intestine. Dig. Dis. Sci. 35: 1505-1517, 1990[Medline].

25.   Rudolphi, K. A., P. Schubert, F. E. Parkinson, and B. B. Fredholm. Adenosine and brain ischemia. Cerebrovasc. Brain Metab. Rev. 4: 346-369, 1992[Medline].

26.   Rudolphi, K. A., P. Schubert, F. E. Parkinson, and B. B. Fredholm. Neuroprotective role of adenosine in cerebral ischaemia. Trends Pharmacol. Sci. 13: 439-445, 1992[Medline].

27.   Sarr, M. G. Motility and absorption in the transplanted gut. Transplant. Proc. 28: 2535-2538, 1996[Medline].

28.   Schubert, P., T. Ogata, C. Marchini, S. Ferroni, and K. Rudolphi. Protective mechanisms of adenosine in neurons and glial cells. Ann. NY Acad. Sci. 825: 1-10, 1997[Abstract].

29.   Thompson, J. S. The intestinal response to critical illness. Am. J. Gastroenterol. 90: 190-200, 1995[Medline].

30.   Van Thiel, D. H., and J. A. Perper. Gastrointestinal complications of cocaine abuse. Recent Dev. Alcohol. 10: 331-334, 1992[Medline].

31.   Van Wylen, D. G., T. S. Park, R. Rubio, and R. M. Berne. Increases in cerebral interstitial fluid adenosine concentration during hypoxia, local potassium infusion, and ischemia. J. Cereb. Blood Flow Metab. 6: 522-528, 1986[Medline].

32.   von Lubitz, D. K., M. F. Carter, M. Beenhakker, R. C. Lin, and K. A. Jacobson. Adenosine: a prototherapeutic concept in neurodegeneration. Ann. NY Acad. Sci. 765: 163-178, 1995[Medline].

33.   Wakefield, A. J., A. M. Sawyerr, A. P. Dhillon, R. M. Pittilo, P. M. Rowles, A. A. Lewis, and R. E. Pounder. Pathogenesis of Crohn's disease: multifocal gastrointestinal infarction. Lancet 2: 1057-1062, 1989[Medline].

34.   Wang, Y. F., Y. K. Mao, J. E. Fox-Threlkeld, T. J. McDonald, and E. E. Daniel. Colocalization of inhibitory mediators, NO, VIP and galanin, in canine enteric nerves. Peptides 19: 99-112, 1998[Medline].

35.   White, T. D., and R. A. Leslie. Depolarization-induced release of adenosine 5'-triphosphate from isolated varicosities derived from the myenteric plexus of the guinea pig small intestine. J. Neurosci. 2: 206-215, 1982[Abstract].

36.   Wiley, J. W., Y. X. Lu, and C. Owyang. Evidence for a glutamatergic neural pathway in the myenteric plexus. Am. J. Physiol. 261 (Gastrointest. Liver Physiol. 24): G693-G700, 1991[Abstract/Free Full Text].

37.   Yano, K., K. Hosokawa, and Y. Hata. Quantitative morphology of Auerbach's plexus in rat intestinal wall undergoing ischemia. J. Reconstr. Microsurg. 13: 297-301, 1997[Medline].


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