Endothelin-mediated vasoconstriction in postischemic newborn intestine

Craig A. Nankervis1, Gail M. Schauer2, and Charles E. Miller3

Departments of 1 Pediatrics and 2 Laboratory Medicine, College of Medicine and Public Health, The Ohio State University and 3 Children's Research Institute, Children's Hospital, Columbus, Ohio 43205


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We previously suggested that the profound, sustained vasoconstriction noted in 3-day-old swine intestine after a moderate episode of ischemia-reperfusion (I/R) reflects the unmasking of underlying constrictor tone consequent to a loss of endothelium-derived nitric oxide (NO). In this study, we sought to determine whether endothelin-1 (ET-1) was the unmasked constrictor and whether selective loss of endothelial ETB receptors, which mediate NO-based vasodilation, participated in the hemodynamic consequences of I/R in newborn intestine. Studies were performed in innervated, autoperfused intestinal loops in 3- and 35-day-old swine. Selective blockade of ETA receptors with BQ-610 had no effect on hemodynamics under control conditions; however, when administered before and during I/R, BQ-610 significantly attenuated the post-I/R vasoconstriction and reduction in arteriovenous O2 difference in the younger group. In 3-day-old intestine, reduction of intestinal O2 uptake to a level similar to that noted after I/R by lowering tissue temperature had no effect on the response to BQ-610 or ET-1, indicating that the change in response to BQ-610 noted after I/R was not simply consequent to the reduction in tissue O2 demand. In studies in mesenteric artery rings suspended in myographs, we observed a leftward shift in the dose-response curve for ET-1 after selective blockade of ETB receptors with BQ-788 in 3- but not 35-day-old swine. Rings exposed to I/R in vivo behaved in a manner similar to control rings treated with BQ-788 or endothelium-denuded non-I/R rings.

swine; intestinal oxygen transport; resistance vessels; precapillary sphincters; ETA receptor; ETB receptor


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

WE PREVIOUSLY REPORTED (16) that intestinal loops from 3- but not 35-day-old swine exposed to a relatively modest degree of ischemia-reperfusion (I/R), i.e., 1 h of flow reduction to 25% of baseline, followed by 2 h of reperfusion, subsequently demonstrated significant and sustained vasoconstriction. These hemodynamic changes were associated with a substantial compromise of intestinal O2 uptake; however, gross inspection of the intestine did not reveal evidence of hemorrhage or overt tissue damage (16). Thereafter, study by our laboratory (14) demonstrated that agonist-induced stimulation of nitric oxide (NO) production was attenuated in mesenteric artery rings exposed to a similar I/R episode and that once again the effect was age specific, occurring to a far greater extent in rings harvested from newborn than from older swine. Two hypotheses were derived from these studies: 1) the initial site of damage after moderate I/R episodes is the intestinal vasculature, primarily the endothelium, and 2) there is a constrictor mechanism present in newborn intestine that is unmasked by the loss of NO-based dilator tone. Thus loss of dilator tone will lead to constriction if, and only if, a constrictor mechanism is present. This constrictor might be constitutively present before the I/R episode; alternatively, its production or efficacy might be increased as a result of the perturbation. The first aim of the present study was to test these hypotheses.

An attractive candidate for the role of post-I/R constrictor is endothelin-1 (ET-1) (28). This peptide is produced in the newborn intestinal endothelium (13), and it has been identified as an important participant in I/R-induced vasoconstriction in other circulations (7, 11, 23, 24, 26). ET-1 is the most biologically active member of a family of isopeptides (8) and exerts its vasoconstrictive effect by binding to the ETA receptor that is primarily located on vascular smooth muscle (1, 3, 4). Ligand binding activates a signal transduction pathway wherein Galpha q/11 activation of phospholipase C causes production of inositol triphosphate and diacylglycerol; these agents increase intracellular calcium concentration and engage the contractile machinery of the cell (21, 22). A second receptor subtype, designated ETB, has been localized to endothelial and vascular smooth muscle cells (6, 11). Activation of the endothelial ETB receptor causes NO-based vasodilation, whereas activation of the vascular smooth muscle ETB receptor causes vasoconstriction (4, 25). Although it is clear that the intestinal vasculature of 3- and 35-day-old swine intestine contract in response to ET-1, the relative presence of ETA and ETB receptors within the postnatal intestinal circulation has not been established. Hence, a second aim of this study was to determine the presence of these receptors in mesenteric artery rings by application of selective agonists and antagonists and to determine if prior exposure to I/R altered these responses.

Three experiments were carried out. First, to determine if ET-1 participates in the vasoconstriction that occurs after a moderate I/R episode in newborn intestine, responses were compared between control animals and those pretreated with the selective ETA receptor antagonist BQ-610. This protocol also included histological evaluation of the intestinal tissue to allow confirmation that the moderate degree of I/R induced in this model was insufficient to cause significant mucosal disruption. In the second experiment, intestinal O2 uptake was lowered by hypothermia, and the effects of this action on the response to BQ-610 and ET-1 were determined. This experiment was carried out to confirm that the enhancement of constrictor tone after I/R was not consequent to reduced tissue O2 demand and subsequent loss of vasodilator tone generated by the metabolic feedback signal (6), which might occur after significant damage to the parenchyma. Finally, the physiological activity of ETA and ETB receptors was compared between mesenteric artery rings harvested from control animals with rings harvested from animals exposed to intestinal I/R in vivo.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Studies were conducted using farm-bred swine of either sex. Two age groups were studied: 3- (newborn; range 2-4 days) and 35-day-old swine (weanling; range 32-38 days). Three-day-old swine are representative of newborn hemodynamic physiology, whereas 35-day-old swine represent preadolescent physiology inasmuch as these older, weanling animals have been entirely weaned from a milk diet (5, 20). All animals were obtained on the day before use and fasted for 12-18 h before surgery. Surgery was carried out under anesthesia with tiletamine hydrochloride/zolazepam hydrochloride (7.5 mg/kg im) and xylazine (5 mg/kg im); the animals were killed by overdose with pentobarbital sodium (6 mg/kg iv). Animal care was provided in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals [DHHS Publication No. (NIH) 85-23, Revised 1985, Office of Science and Health Reports, Bethesda, MD 20892], and all experimental protocols were approved and monitored by the Children's Hospital Institutional Animal Care and Use Committee.

In Vivo Isolated Gut Loop Studies

Experimental preparation. Animals were anesthetized, an airway was established, and ventilation was begun to maintain normal blood gas tensions. A femoral artery-vein pair was cannulated to monitor blood pressure, heart rate, and blood gases, infuse 5% dextrose in 0.9% saline (10 ml · kg-1 · min-1), administer additional anesthetic, and return blood. A loop of distal jejunum-proximal ileum that was ~25 cm long and served by a single artery-vein pair was isolated from the remainder of the gut and cleansed by repetitive luminal instillation of warm saline and air. The periarterial nerve bundle was left intact. Heparin was administered (500 U/kg), and the mesenteric vein draining the segment was cannulated. This catheter was directed through an electromagnetic flowmeter and pressure transducer and then to a venous collection reservoir primed with blood from a previous study. Blood was pumped from the venous reservoir back to the animal via the femoral vein catheter at a rate set to match venous outflow from the animal. Venous pressure was set and maintained at 0 mmHg by adjusting the height of the catheter with respect to the gut loop. A small-bore (27-gauge) needle was inserted into the portion of the mesenteric artery perfusing the isolated gut loop to allow drug infusion. The mesenteric artery line was kept patent by infusing 0.9% saline at 0.1 ml/h.

In animals used in the I/R protocols, the viscera, including the isolated gut loop, were returned to the peritoneal cavity, which was then closed. The animal was then placed on the right side, and a left flank incision was made, exposing the root of the mesenteric artery at its origin from the abdominal aorta. The mesenteric artery was encircled by a silk tie. A plastic cylinder (outer diameter, 3 mm for 3-day-old swine and 6 mm for 35-day-old swine) was placed between the tie and the artery, the tie was tightened, and the cylinder was removed. This action created a coarctation in the mesenteric artery that reduced the flow rate to the isolated gut loop by ~75%. The extent of flow reduction was measured in each animal throughout the duration of the ischemic period. The coarctation was relieved by removing the tie. In control animals, the tie was placed in a similar fashion but not tightened. Throughout the course of the experiment, the temperature of each animal was monitored by a rectal thermistor attached to a servo device that regulated a heating pad placed beneath the animal, which was also covered to minimize heat loss. Rectal temperature was maintained at 37°C.

In animals used in the hypothermia studies, the isolated intestinal loop was left exteriorized and was separated from the abdominal incision by several layers of gauze. The tissue was covered with plastic film to minimize evaporative heat loss. A second thermistor was placed into the lumen of the intestinal loop and attached to a servo device that regulated an overhead infrared lamp. Reduction of the setpoint on this servo device led to a rapid reduction in the temperature of the intestinal loop, without concomitant change in the core temperature of the animal. The temperature within the intestinal lumen was maintained at 32°C.

Measurement techniques. Vascular pressures were measured with standard transducers (model P23ID, Gould Statham, Oxnard, CA) attached via T connectors to the femoral artery and mesenteric vein catheters and calibrated with a mercury manometer. Blood flow was measured with an electromagnetic flowmeter (model 2200, Gould; 1.5 mm inside diameter); zero flow was determined by complete occlusion of the venous circuit distal to the flowmeter. Blood gas tensions and O2 contents were measured using a standard gas analyzer (model 168, Corning Medical, Medfield, MA) and Lex-O2-Con (Hospex, Chestnut Hill, MA), respectively.

Experimental protocols. Four protocols were carried out. In protocol 1, hemodynamic and oxygenation parameters were measured under baseline conditions, during 1 h of ischemia (75% flow reduction) and during 2 h of reperfusion. Controls for this protocol were followed for 3 h, without creation of I/R. At the completion of the protocol, segments of isolated gut were fixed in 10% Formalin and later processed for light microscopy. In protocol 2, a continuous infusion of BQ-610 into the mesenteric artery catheter (0.1 ml/h, providing 10-6 M/h) was initiated after collection of baseline data; when new steady-state conditions were attained, an episode of I/R was created in a manner identical to that described for protocol 1. Controls for this protocol received an identical infusion of BQ-610 for 3 h, without creation of I/R. In protocol 3, intestinal temperature was reduced from 37°C to 32°C after collection of baseline data; once new steady-state conditions were attained, a single bolus dose of ET-1 (10-9 M/kg) was given into the mesenteric artery. Controls for this protocol received ET-1, but were not made hypothermic. In protocol 4, a continuous infusion of BQ-610 was started into the mesenteric artery in a manner identical to that previously described, except that intestinal hypothermia was created after postdrug steady-state hemodynamics were attained instead of I/R.

Data analysis. To carry out the histological analysis, four slides were prepared from each animal used in protocol 1; both control animals and those exposed to I/R. The slides were overlabeled with random numbers and assessed by a pathologist unaware of the age group or treatment of each specimen. Histological findings were assessed according to a scoring system for ischemic intestinal damage in swine outlined by Cheung and colleagues (2). Calculation of O2 delivery and uptake were carried out using the Fick equation. Evaluation of hemodynamic data was achieved using an ANOVA technique for repetitive measures. Initially, a three-way ANOVA was carried out that utilized age group (3- vs. 35-day-old swine), condition (control vs. I/R vs. hypothermia), and time (baseline, ischemia, and reperfusion) as main effects. If the F statistic for this ANOVA was significant (P < 0.01), subsequent post-hoc Tukey B tests were carried out to determine the sites of significance.

In Vitro Ring Tension Studies

Tissue preparation. I/R was carried out under in vivo conditions, in a manner identical to that described for protocol 1. At the completion of the 1-h ischemia and 2-h reperfusion period, or 3-h observation period for control animals, the entire mesenteric artery was removed en bloc and placed in iced Krebs buffer. The vessel was cleansed of adherent connective tissue and cut into 3-mm rings with care taken to avoid stretching of the vessel or contact with the intimal surface. Rings were cut from the main mesenteric artery trunk between the colic and the first jejunal branch points. In some experiments, mechanical removal of the endothelium was deliberately achieved by passing a coarse silk thread through the vessel lumen three times.

Experimental apparatus. Rings were mounted between two stainless steel stirrups placed within water-jacketed 20-ml glass wells. One stirrup was fixed to the bottom of the well, and the second was tethered to a force transducer (model FT03, Grass Instruments, Quincy, MA) to facilitate continuous measurement of isometric tension on a multichannel recording device (model 7 polygraph, Grass). The well was filled with Krebs buffer of the following composition (in mM): 118.1 NaCl, 4.8 KCl, 2.5 CaCl2, 1.2 Mg SO4, 1.2 KH2PO4, 25.0 NaHCO3, 11.1 glucose, and 0.026 EDTA. The buffer was maintained at 37°C and continuously aerated with 95% O2-5% CO2.

Contractile responses. Rings were progressively stretched over 1-2 h to the optimal point on their length-tension curve, as determined by noting the maximal contractile response to Krebs buffer containing 40 mM KCl. Rings were then allowed to equilibrate for 1 h in fresh buffer at this tension. Endothelial integrity was determined in all rings by noting their response to 10-7 M ACh after precontraction with phenylephrine. Relaxation of >40% of precontraction baseline was accepted as evidence of endothelial integrity, whereas dilation <5%, or any degree of contraction was accepted as evidence of endothelial disruption. In control rings, dose-response curves to 10-11-10-7M ET-1 and 10-11-10-7M sarafotoxin 6c (S6c) were determined by cumulative addition of each agent to selected wells. S6c is a snake venom toxin that shares significant sequence homology to ET-1 and related peptides. It functions as a selective agonist for the ETB receptor and is therefore useful in distinguishing between receptor subtypes in physiological studies (27). Each ring was exposed to only one of the above agents. In some studies, ET-1 and S6c were applied after administration of 10-6 M BQ-610 or BQ-788, which were given to antagonize the ETA or ETB receptor, respectively (9). In I/R and endothelium-denuded non-I/R rings (E-), dose-response curves to 10-11-10-7 M ET-1 were determined by cumulative addition of this agent to selected wells.

Relaxation responses. After determination of the optimal resting tension and endothelial integrity, the rings were precontracted to 75% of their maximal tension with phenylephrine. A dose-response curve to 10-11-10-7 M S6c was determined in control rings by cumulative addition of this agent to selected wells. In some studies, S6c was applied after the administration of 10-6 M BQ-610 or BQ-788, which were given to antagonize the ETA or ETB receptor, respectively, or 10-4 M NG-monomethyl-L-arginine (L-NMMA) given to block NO synthase (18). In I/R rings and E- rings, a dose-response curve to 10-11-10-7 M S6c was determined by cumulative addition of this agent to selected wells.

Data analysis. All observations were made in paired rings, and the average response was determined. In statistical analysis and data presentation, n refers to the number of animals in which observations were made. For the contractile responses, absolute changes in developed tension were recorded in grams, but were expressed as a percentage of the maximal contractile response to 40 mM KCl to facilitate comparison between age groups. This transformation proved necessary inasmuch as the resting tension and magnitude of change were consistently greater in rings from 35-day-old swine because their internal and external diameters were substantially greater. For the relaxation responses, absolute changes in developed tension were recorded in grams but were expressed as percent relaxation of the phenylephrine-induced precontraction. Responses were compared between age groups by a three-way ANOVA, which utilized group (3- vs. 35-day-old swine), condition (control vs. I/R vs. E-), and drug concentration as main effects. Post-hoc Tukey B tests were carried out on data sets in which the F statistic for ANOVA was significant (P < 0.01) to determine the sites of significant differences between variables.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In Vivo Isolated Gut Loop Studies

Baseline hemodynamic and oxygenation variables were age specific (Table 1). Creation of the mesenteric artery coarctation reduced intestinal blood flow ~75%; thus flow into the isolated gut loop fell from 16 ± 2 to 4 ± 1 ml/min in the 3-day-old group and from 17 ± 3 to 5 ± 1 ml/min in the 35-day-old group. The average tissue weights for the isolated gut loops were 17.2 ± 1.2 and 36.8 ± 3.4 g in 3- and 35-day-old swine, respectively.

                              
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Table 1.   Baseline hemodynamic and oxygenation variables for in vivo preparations

Changes in hemodynamic and oxygenation variables during reperfusion were significantly age specific (Figs. 1-3). Younger animals demonstrated an initial hyperemia 5 min into reperfusion; this observed hyperemia was short lived, however, as vascular resistance rapidly increased to a level significantly above baseline 15 min into reperfusion. Consequent to this progressive vasoconstriction, intestinal blood flow, and thus O2 delivery fell during reperfusion so that these variables were 35% lower than baseline at the completion of the reperfusion period. Arteriovenous O2 difference [(a-v)O2] increased 5 min into reperfusion, but then decreased to a level below baseline as reperfusion continued. Intestinal O2 uptake was reduced to 55% of its baseline level at the completion of the 2-h reperfusion period. In stark contrast to 3-day-old swine, 35-day-old swine demonstrated a sustained vasodilation during reperfusion. This action caused O2 delivery to increase during reperfusion. (a-v)O2 initially rose, but then fell during reperfusion, so that tissue O2 uptake remained near baseline throughout the reperfusion period, once the initial postischemic hyperemia abated. Control animals demonstrated no significant change in any measured variable over 3 h (data not shown).


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Fig. 1.   The effects of BQ-610 infusion on intestinal vascular resistance within in vivo segments of small intestine from 3- and 35-day-old swine after 1 h of partial (75%) ischemia and 2 h of reperfusion. , Control; open circle , BQ-610-treated intestine. B, baseline. Data are means ± SD; n = 5 for both age groups. a P < 0.01 vs. control. Note that BQ-610 infusion was started at the onset of reperfusion and continued throughout the reperfusion period.



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Fig. 2.   O2 delivery, arteriovenous O2 difference [(a-v)O2], and O2 uptake within in vivo segments of small intestine from 3-day-old swine after 1 h of partial (75%) ischemia and 2 h of reperfusion. , Control; open circle , BQ-610-treated intestine. Data are means ± SD; n = 5 for both groups. a P < 0.01 vs. control.



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Fig. 3.   O2 delivery, (a-v)O2, and O2 uptake within in vivo segments of small intestine from 35-day-old swine after 1 h of partial (75%) ischemia and 2 h of reperfusion. , Control; open circle , BQ-610-treated intestine. Data are means ± SD; n = 5 for both groups.

Continuous infusion of the selective ETA receptor antagonist BQ-610 to control animals had no effect on intestinal vascular resistance; however, (a-v)O2 and O2 uptake were increased with respect to baseline once steady-state conditions were attained during drug infusion (Table 2). The effects of BQ-610 on hemodynamic and oxygenation variables during reperfusion were age specific (Figs. 1-3). In the 3-day-old group, BQ-610 significantly attenuated the increase in vascular resistance noted during reperfusion. This effect on vascular resistance caused blood flow and thus O2 delivery to be less compromised during reperfusion. Also, the reduction in (a-v)O2 noted during reperfusion was significantly diminished in 3-day-old swine administered BQ-610. In the 35-day-old group, BQ-610 had a minimal effect on hemodynamic and oxygenation variables during reperfusion.

                              
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Table 2.   Effects of BQ-610 on hemodynamic and oxygenation variables in postnatal swine intestine

The degree of intestinal damage noted during histological analysis after 1 h of 75% ischemia and 2 h of reperfusion was similarly mild in both age groups. In 3-day-old intestine, mean injury scores of 0.08 ± 0.4 and 1.59 ± 0.22 were noted in the control and I/R subgroups (with a score of 6 indicating the most severe damage), respectively. In 35-day-old intestine, mean injury scores were 0.00 and 1.15 + 0.12 in the control and I/R subgroups, respectively.

Reduction of intestinal temperature from 37°C to 32°C decreased tissue O2 uptake by ~40%. This reduction was reflected by concomitant decreases in blood flow and thus O2 delivery, as well as in the percentage of O2 extracted by the intestine, as evidenced by a decline in (a-v)O2 (Table 3). However, hypothermic intestine demonstrated vasoconstriction in response to a bolus injection of ET-1 that was similar in magnitude to that noted in control, i.e., normothermic intestine. ET-1 exerted a constrictor effect on both resistance vessels, as evidenced by an increase in intestinal vascular resistance, and on exchange vessels (precapillary sphincters), as noted by a reduction in (a-v)O2. Pretreatment with BQ-610 had no effect on the response of the intestine to hypothermia (Table 4).

                              
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Table 3.   Effect of ET-1 administration on intestinal hemodynamics and oxygenation in 3-day-old swine


                              
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Table 4.   Effects of BQ-610 on intestinal hemodynamics and oxygenation in 3-day-old swine with hypothermia-depressed intestinal O2 uptake

In Vitro Ring Tension Studies

Contractile responses. Application of ET-1 to control rings caused similar concentration-dependent increases in developed tension in both age groups. Pretreatment with BQ-788 significantly increased the contractions noted in response to ET-1 in younger animals, but had no effect on the response to this peptide in older animals (Fig. 4). A similar leftward shift in the dose-response curve for ET-1 was noted in E- rings in newborn but not weanling swine. The response of mesenteric artery rings exposed to I/R in vivo was statistically similar to that of control rings treated with BQ-788 or of E- control rings in newborn swine. Thus exposure to I/R caused a significant leftward shift of the dose-response curve for ET-1 in newborn swine. Exposure to I/R caused a significant rightward shift of the dose-response curve for ET-1 in older swine. In all cases, contractions due to ET-1 administration were significantly attenuated by prior blockade of the ETA receptor with BQ-610, whereas S6c failed to induce contraction in quiescent rings from either age group (data not shown).


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Fig. 4.   The effects of endothelin-1 (ET-1) administration on developed tension within in vitro segments of relaxed mesenteric artery from 3 (A)- and 35 (B)-day-old swine. , Control; open circle , post-ischemia-reperfusion (post-I/R); triangle , BQ-788; , endothelium-denuded non-I/R rings. Data are means ± SE; n = 5 for both age groups. a P < 0.01 vs. control.

Relaxation responses. Application of S6c to control rings caused concentration-dependent reduction in developed tension that was significantly greater in younger swine. Pretreatment with BQ-788 or L-NMMA significantly decreased the dilations noted in response to S6c in both age groups (Fig. 5). S6c-mediated relaxation was significantly attenuated in rings exposed to I/R in vivo, before in vitro analysis. This effect was significantly less pronounced in rings obtained from weanling animals exposed to I/R in vivo. In all cases, dilations due to S6c administration were not significantly attenuated by prior blockade of the ETA receptor with BQ-610, whereas S6c failed to induce relaxation in precontracted E- rings from either age group (data not shown).


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Fig. 5.   The effects of sarafotoxin 6c (S6c) administration on developed tension within in vivo segments of phenylephrine (PE)-precontracted mesenteric artery from 3 (A)- and 35 (B)-day-old swine. , Control; open circle , post-I/R; triangle , BQ-788; , NG-monomethyl-L-arginine. Data are means ± SE; n = 5 for both age groups. a P < 0.01 vs. control; b P < 0.01 vs. 3-day-old control.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Two novel observations were made in this study. First, the vasoconstriction that follows a moderate I/R episode in newborn intestine is mediated, in part, by ET-1; moreover, the depression of newborn intestinal O2 uptake after I/R reflects ET-1-induced impairment of O2 transport and not a decline in O2 demand caused by wide-spread damage to the intestinal parenchyma. Second, ETB receptors are present on the endothelium within the postnatal intestinal vasculature, and their function is age specific, being more substantial in younger intestine. Furthermore, loss of endothelial ETB receptors after a moderate I/R episode appears to occur in newborn intestine and may explain, in part, the increased vascular tone noted after I/R in these animals.

ET Participates in Post-I/R Vasoconstriction in Newborn Intestine

The effect of ETA receptor blockade on intestinal hemodynamics clearly changed after I/R in 3- but not 35-day-old intestine, indicating that ET-1 is responsible, at least in part, for the contraction noted in newborn intestine under these circumstances. We are unable to conclude, however, if the local production or the contractile efficacy of ET-1 increased after I/R or if the role of existing peptide was simply amplified by the loss of the counterbalancing NO-based dilation. We speculate that the former supposition is correct, based on two lines of reasoning. First, increased local production of ET-1 has been demonstrated after I/R events in other circulations (7, 26) and in cardiac myocytes (24); furthermore, the endothelium of newborn intestine is capable of producing ET-1 (13). Second, it is possible that the binding affinity of the ETA receptor increased after I/R consequent to a reduction of NO within the microcirculatory environment. NO interferes with G protein-receptor interaction, reducing the binding affinity of the receptor for its ligand (12, 17, 19). A loss of NO, which we (13) have demonstrated in post-I/R 3-day-old intestine, might thus enhance the coupling of the ETA receptor with its G protein, increasing receptor affinity for ET-1 very quickly. This action could amplify the constrictor effect of existing peptide without the need for generation of new peptide or receptor in response to the I/R insult.

The depression of newborn intestinal O2 uptake after a moderate I/R episode could reflect two events: 1) reduction of O2 demand secondary to tissue damage or 2) impairment of O2 transport. We interpret the present observations as supportive of the latter possibility. Histological evidence of tissue damage was minimal. A complete morphometric evaluation, particularly demonstrating intact mitochondria might provide more reassuring visual evidence that parenchymal elements were not significantly affected by the moderate I/R episode imposed in these experiments. Alternatively, direct measurement of O2 utilization by the intestine, instead of indirect estimation of uptake based on the Fick principle, would obviously provide definitive proof that intestinal O2 demand was not decreased after I/R. It remains, however, that the tissue appeared unaffected by the I/R episode; indeed, it most likely that O2 demand was increased in 3-day-old intestine after I/R, a circumstance that was clearly present in older animals. Hypothermia-induced depression of intestinal O2 uptake was not mediated by ET-1, as evidenced by the lack of effect of BQ-610 on the response to hypothermia. This is an important observation, as loss of dilator tone generated by a reduction of the metabolic feedback signal (6) could enhance the response to constrictor agents, providing an alternative explanation for the enhanced role of ET-1 after I/R. Also, the constriction generated by application of exogenous ET-1 confirms that hypothermia did not compromise the contractile machinery of the intestinal vasculature, an observation that validates the hypothermia model as a means to reduce intestinal O2 demand without affecting O2 transport. These data support our contention that the post-I/R reduction in newborn intestinal O2 uptake occurs because of disruption of O2 transport, rather than reduction of tissue O2 demand. These data also support our hypothesis that the principal target of moderate I/R episodes in newborn intestine is its vasculature, which then becomes relatively incapable of supporting the oxygenation requirements of the parenchyma, leading to sustained tissue hypoxia.

It is most likely that the post-I/R disruption of O2 transport occurred at both resistance vessels and exchange vessels within the intestinal microcirculation. The effect on resistance vessel function is easily determined by noting the ET-1-mediated increase in vascular resistance and thus reduction of blood flow and O2 delivery to the intestine (6). The effect on exchange vessels, or more precisely, precapillary sphincters is evidenced by the rise of mesenteric vein O2 partial pressure (PO2) and reduction in (a-v)O2 after I/R. Under normal circumstances, a fall in O2 delivery would reduce capillary PO2 if tissue O2 demand, capillary surface area, and the capillary-to-cell distance remain constant, because O2 would continue to diffuse from capillary to cell without sufficient replacement in the vascular space by incoming red blood cells (6). Also, a reduction in O2 delivery increases net capillary perfusion in newborn intestine, thus decreasing (a-v)O2 as a greater percentage of the delivered O2 is extracted (15). Here, we observed the opposite effects and contend that they reflect the effect of ET-1 on precapillary sphincters, specifically causing their closure and thus reducing the surface area available of O2 diffusion, as well as increasing the capillary-to-cell distance. This speculation is supported by our (6, 10) earlier report that the capillary filtration coefficient, a marker of capillary surface area, is reduced by exogenous ET-1 in newborn intestine (18).

Functional Activity of ETA and ETB Receptors in Postnatal Intestine

Both 3- and 35-day-old intestine possess functional ETA receptors that mediate pronounced and sustained vasoconstriction in response to ligand binding (13). The novel observation of this study is that ETB receptors are present within the newborn intestinal vasculature, that they are located primarily on the vascular endothelium, and that their presence is developmentally regulated. Evidence to this effect includes the enhancement of the contractile effect of ET-1 in the presence of selective ETB receptor blockade; thus mesenteric artery rings from 3- but not from 35-day-old swine demonstrated a significant leftward shift in the ET-1 dose-response curve after administration of BQ-788 and this shift was duplicated in E- rings. Also, precontracted rings from 3- but not 35-day-old swine demonstrated a pronounced relaxation in response to the ETB-selective agonist S6c that was blocked by BQ-788 or L-NMMA.

The presence of endothelial ETB receptors in newborn intestinal circulation may have contributed to the hemodynamic changes noted after I/R in this circulation. The presence of endothelial ETB receptors whose activation leads to NO-based vasodilation acts to counterbalance ETA receptor-induced vasoconstriction; this interaction was evident in the leftward shift of the ET-1 dose-response curve after selective ETB receptor blockade with BQ-788. We (13) have previously demonstrated that moderate I/R episodes disrupt endothelial function in newborn intestine. One aspect of this disruption could be the relative loss of ETB receptor function. This possibility is supported by the leftward shift of the ET-1 dose-response curve in mesenteric artery rings from 3-day-old swine previously exposed to I/R.

In summary, a moderate episode of I/R, i.e., flow reduction to 25% of baseline for 1 h followed by 2 h of reperfusion, causes vasoconstriction and reduction of (a-v)O2 in 3-day-old intestine. These hemodynamic changes are mediated, in part, by ET-1 and result in significant compromise of O2 transport, causing depression of O2 uptake. They are also age specific, as they do not occur in 35-day-old intestine. One reason for this age specificity may be a greater functional presence of endothelial ETB receptors in newborn intestine and loss of these receptors consequent to I/R-induced endothelial cell damage.


    ACKNOWLEDGEMENTS

We thank Dr. Philip Nowicki for review of the manuscript and Karen Bentley for secretarial assistance.


    FOOTNOTES

This work was supported by Children's Research Institute Grant 213798.

Address for reprint requests and other correspondence: C. A. Nankervis, Children's Hospital, 700 Children's Dr., Columbus, OH 43205 (E-mail: nankervisc{at}pediatrics.ohio-state.edu).

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 26 July 1999; accepted in final form 6 April 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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