Effects of systemic hypotension on postnatal intestinal circulation: role of angiotensin

Philip T. Nowicki and Lisa A. Minnich

Departments of Pediatrics and Physiology, The Ohio State University and The Wexner Institute of Pediatric Research, Columbus, Ohio 43205


    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Systemic hypotension causes a greater degree of vasoconstriction in intestine from 3- than from 35-day-old postnatal swine. To determine the basis for this age-dependent difference, systemic hypotension (pressure reduction to ~50% of baseline) was induced by creating pericardial tamponade in postnatal swine instrumented to allow measurement of intestinal hemodynamics and oxygenation in vivo. Hypotension caused gut vascular resistance to increase 77 ± 6% in 3-day-old subjects but only 18 ± 3% in 35-day-old subjects. Prior blockade of alpha 1-receptors with phentolamine, vasopressin receptors with [d(CH2)5,D-Phe2,Ile4,Ala9-NH2]AVP, or surgical denervation of the gut loop had no effect on hypotension-induced gut vasoconstriction. Losartan, which blocks angiotensin AT1 receptors, significantly attenuated hypotension-induced gut vasoconstriction in both age groups. BQ-610, which blocks endothelin ETA receptors, also limited the magnitude of vasoconstriction but only in younger subjects. This effect may have been consequent to an interaction between endothelin and angiotensin, inasmuch as a subpressor concentration of endothelin increased the contractile response to angiotensin in mesenteric artery rings. The substantial rise in 3-day-old gut vascular resistance was partly consequent to a locally mediated vasoconstriction that occurred in response to pressure and/or flow reduction during hypotension, as evidenced by the significant attenuation of this constriction when blood flow was held constant by controlled-flow perfusion to the gut loop during hypotension. Intestinal O2 uptake was compromised to a significantly greater degree in 3- than in 35-day-old subjects during hypotension. This difference was primarily due to the inability of younger intestine to increase O2 extraction in the face of reduced blood flow and may be mediated, in part, by an effect of angiotensin II on intestinal capillary perfusion.

newborn; nitric oxide; endothelium; endothelin


    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

HYPOTENSION ELICITS a well-characterized release of vasoconstrictor stimuli designed to restore systemic arterial pressure by enhancing peripheral vascular tone, increasing inotropy and heart rate, and augmenting circulating blood volume (13). Baroreceptor- and then chemoreceptor-initiated neural responses occur first, followed by increased circulating concentrations of catecholamines, angiotensin (ANG), vasopressin (VP), and endothelin (ET). An important target of these constrictor stimuli is the splanchnic circulation; thus, constriction of mesenteric capacitance vessels increases circulating blood volume, whereas increased mesenteric arterial resistance raises systemic blood pressure and redirects available cardiac output elsewhere. The deleterious effects of this systemic pressor response on intestinal oxygenation are attenuated by a locally mediated vasodilation termed autoregulatory escape (16), which partially restores the convective phase of O2 transport, and by expansion of the perfused capillary density (16), which augments the diffusive phase of O2 transport (12).

The ability of the newborn intestinal circulation to engage in locally mediated autoregulatory escape from the systemic pressor response is limited (5, 25). Thus, although escape from sustained mesenteric nerve stimulation is intact in newborn intestine (25), the vasodilator compensation to sustained infusion of phenylephrine (PE), ANG II, or VP is deficient (23); furthermore, in vitro segments of newborn intestine demonstrate significant vasoconstriction in response to reduced perfusion pressure (22, 24). Together, these circumstances lead to marked vasoconstriction within newborn intestine during systemic hypotension resulting from pericardial tamponade (23) or hemorrhage (30). While it is clear that newborn intestinal perfusion is reduced during systemic hypotension, it has yet to be determined which systemic pressor system is primarily responsible for this effect. Furthermore, the relative contribution of intrinsic or locally mediated vascular effector systems to the intestinal vasoconstriction that occurs during systemic hypotension has not been established.

The goal of this work was to more clearly delineate the mechanistic basis for newborn intestinal vasoconstriction during systemic hypotension. To this end, systemic hypotension was induced by means of the cardiac tamponade model of Bulkley and colleagues (6, 7) in the presence of selective blockade of alpha 1-receptors, ANG AT1 receptors, ETA receptors, and VP receptors; as well, studies were carried out in surgically denervated intestine. The contribution of the local intestinal response to flow and pressure reduction that accompanied systemic hypotension was determined by holding these variables constant within an isolated intestinal loop during tamponade. In all instances, comparisons were made between the newborn response, i.e., in 3-day-old swine, with that noted in older subjects, i.e., 35-day-old swine, to determine if these responses were age dependent.


    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Animal Acquisition and Handling

Studies were conducted on postnatal swine of two ages: 3-days old (range 2-4 days) and 35-days old (range 33-37 days). Subjects were obtained from a local breeding farm on the day before use and were fasted for 8-12 h before surgery. Anesthesia was induced with tiletamine hydrochloride-zolazepam hydrochloride (6 mg/kg im) and xylazine (4 mg/kg im) and was maintained with pentobarbital sodium (5 mg/kg iv given every 60 min or sooner if deemed necessary by the vivarium staff). Animals were killed by an overdose of pentobarbital sodium while still anesthetized. All animal care was provided in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, publication no. 85-23) under the auspices of an experimental protocol approved by the Children's Hospital Research Foundation Institutional Animal Care and Use Committee. All work was carried out in the Wexner Institute vivarium, an American Association of Accreditation of Laboratory Animal Care-approved facility, under the supervision of a veterinarian.

Experimental Preparations

Measurement of isometric tension in mesenteric artery rings in vitro. The mesenteric artery trunk was removed en bloc and was placed in Krebs buffer on a dissecting tray positioned over ice. Rings (3 mm) were cut with care taken to avoid contact with the intimal surface of the vessel and were mounted between two stainless steel stirrups placed within water-jacketed 20-ml glass myographs. One stirrup was tethered to a force transducer (Grass Instruments FT-03, Quincy, MA) to allow measurement of isometric tension on a multichannel recording device. The well was filled with Krebs buffer of the following composition (in mM): 118.1 NaCl, 4.8 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25.0 NaHCO3, 11.1 glucose, and 0.026 EDTA. The buffer was maintained at 38°C and was continuously aerated with 95% O2-5% CO2. Rings were stretched progressively over 1-2 h to the optimal point on their length-tension curve, as determined by noting the maximal contractile response to 80 mM KCl Krebs buffer. Endothelial integrity was determined in all rings by noting >50% dilation of PE-precontracted rings to acetylcholine (10-7 M).

Measurement of hemodynamics and oxygenation within in vivo intestine. Subjects were anesthetized and ventilated to maintain normal blood gas tensions. A femoral artery-vein pair was cannulated; the arterial cannula was directed to a pressure transducer, whereas the venous cannula served as a delivery site for crystalloid (5% dextose in 0.9% saline, 15 ml · kg-1 · h-1) and also for blood return. A segment of distal jejunum-proximal ileum ~25 cm long was isolated vascularly from the remainder of the gut so that it was perfused and drained by a single artery-vein pair. The animal was administered heparin, 500 U/kg, and then the vein was cannulated, and the catheter was directed to a beaker primed with 50 ml of heparinized swine blood. A flowmeter (2.0 mm ID; Gould, Cleveland, OH) was placed in the venous circuit to allow flow measurement. Blood collected in the beaker was pumped back into the subject at a rate equal to venous outflow, and the venous pressure was kept at 0 mmHg by adjusting the height of the venous catheter with respect to the animal. A narrow (25-gauge) needle was inserted in the mesenteric artery perfusing the isolated gut loop; this insertion was carried out without prior dissection of the artery so as to minimize damage to the periarterial nerves. The isolated gut loop and abdominal wound were covered by thin plastic film to minimize evaporative heat and water loss. Temperature probes placed in the rectum and within the lumen of the isolated gut loop were connected to a servocontrolled heating pad placed under the animal and to an overhead radiant heating element, respectively, to keep temperature at 38°C. The pericardium was exposed by a left-sided thoracic incision between the fifth and sixth ribs. A soft plastic catheter was placed in the pericardial sac via a small puncture wound, which was thereafter repaired, restoring the integrity of the pericardial cavity. The thoracic wound was closed with silk sutures, and the left lung was reinflated. Total blood O2 content and blood gas tensions were determined in paired arterial-mesenteric vein blood samples (Lex-O2-Con; Chestnut Hill, MA, and Corning Blood Gas Analyzer). To assure that blood O2 contents had reached steady-state levels before sampling, blood PO2 was sampled two times, at 1-min intervals, before samples for O2 content were obtained.

Experimental Protocols

Protocol I: Determination of the IC50 of selected blocking agents. It was first necessary to determine the effective blocking concentrations of the selected receptor antagonists. To this end, the IC50 for each antagonist was determined using in vitro mesenteric artery rings, and then the value was confirmed in vivo. To begin, the ED50, i.e., the agonist concentration that resulted in 50% of the maximal contraction of each ring to 80 mM KCl, was determined for PE, ANG II, VP, and ET-1 by administering progressively increasing concentrations of these agonists to relaxed mesenteric artery rings. The dose ranges used were as follows: PE, 10-8 to 10-4 M; ANG II, 10-10 to 10-6 M; VP, 10-11 to 10-8 M; and ET-1, 10-10 to 10-6 M. All observations were made in paired rings from three subjects of both age groups, and each ring was administered only one agonist. Thereafter, the IC50, or concentration of receptor antagonist that reduced the contractile response noted in response to the respective agonist by 50%, was determined by administering the ED50 for each agonist to rings pretreated with serial dilutions of the appropriate antagonist (19). The following blocking agents were used: phentolamine (10-8 to 10-6 M) to block alpha 1-receptors (21); losartan (10-8 to 10-6 M) to block ANG AT1 receptors (28); [d(CH2)5,D-Phe2,Ile4,Ala9-NH2]AVP (PIA, 10-9 to 10-7 M) to block VP receptors (18); and BQ-610 (10-8 to 10-6 M) to block ETA receptors (all agents from Peninsula Laboratories, Belmont, CA; see Ref. 8). This protocol was carried out in paired rings from four subjects from each age group.

Protocol II: Confirmation of blocking doses in vivo. Gut loops were prepared as described, except that the pericardial catheter was not placed. Six subjects were used for each agonist-antagonist pair in each age group; three received a sustained infusion of contractile agonist in the mesenteric artery perfusing the gut loop until steady-state conditions were attained, whereas the remaining three received the same infusion 5 min after intra-arterial infusion of the respective blocking agent. This approach was chosen to eliminate the possibility of tachyphylaxis to a second agonist dose given to a single subject and also because some agonists (e.g., ET-1) cause a sustained contraction that cannot be washed out. The agonist infusion rate was determined as the ED50 for each agent per minute as follows: PE, 8 × 10-6 M/min; ANG II, 9 × 10-9 M/min; VP, 2 × 10-10 M/min; and ET-1, 6 × 10-9 M/min. All agonists were diluted in 0.9% saline and prepared so that the infusion rate was 0.5 ml/min, a rate that did not alter intestinal hemodynamics during saline infusion. The blocking dose of antagonists was chosen as one order of magnitude greater than the IC50, inasmuch as it was desired to attenuate the contractile response by >90% (19).

Protocol III: Induction of systemic hypotension by tamponade with or without blocking agents. Steady-state conditions were achieved, defined as fluctuation of systemic arterial pressure and intestinal blood flow <5% over 10 min. Baseline arterial and mesenteric venous blood samples (0.5 ml each) were taken for blood gas and O2 content analysis, and then a blocking agent or placebo (0.9% saline) was infused in the mesenteric artery. A second, postdrug set of blood samples was taken. Thereafter, a pericardial tamponade was induced by infusing warm (38°C) 10% dextran in the pericardial sac until mean arterial pressure was reduced to ~50% of baseline. Pressure was kept at this level for 30 min; if necessary, additional 10% dextran was infused into, or removed from, the pericardial sac to maintain mean arterial pressure within ±2% of the new, lower level. Repeat blood samples were obtained 5, 15, and 30 min after induction of tamponade. An additional 2 ml of arterial blood were obtained during the initial baseline and 30-min sampling points to measure plasma ANG II and ET-1 levels. The total blood volume removed during the entire protocol was ~10 ml. In 3-day-old subjects, this blood loss was replaced by an equal amount of stored donor swine blood at the completion of each sampling. This replacement was necessary to avoid volume depletion, which might exacerbate the tamponade-induced hypotension, inasmuch as the circulating blood volume at this age is ~80 ml/kg (9), and these subjects weighed 1.8 ± 0.1 kg. The identical protocol was also carried out in some 3- and 35-day-old subjects after surgical denervation of the isolated gut loop. In these subjects, a femoral-mesenteric artery bridge was created by cannulating the second femoral artery and directing this catheter to the mesenteric artery perfusing the isolated gut loop. Thereafter, all mesenteric connections between the isolated gut loop and study subject were severed, assuring complete denervation of the gut loop. This additional manipulation was carried out before beginning the protocol, i.e., before the first baseline measurement. Five subjects were studied in each age group for each condition (i.e., control, blocking agents, denervation).

Protocol IV: Induction of systemic hypotension by tamponade under controlled-flow conditions. Reduction of perfusion pressure causes significant vasoconstriction within in vitro gut loops from 3-day-old swine (24); thus, it was anticipated that some of the constrictor response to tamponade would be consequent to this intrinsic response to pressure reduction. To delineate the constrictor effects of systemic pressors released consequent to tamponade-induced hypotension from the intrinsically mediated vasoconstriction that would occur as a result of reduced perfusion pressure per se, an additional protocol was carried out in which perfusion of the isolated gut loop was maintained during tamponade by means of a femoral-mesenteric artery bridge similar to that just described, with two differences. First, the mesenteric artery was cannulated with care taken to avoid trauma to the periarterial mesenteric nerve fibers, i.e., the isolated gut loop was not severed from the study subject, so that it was not denervated. Second, the femoral-mesenteric artery bridge was drawn though a peristaltic pump (Gilson). To this end, flow rate into the gut loop via the femoral-mesenteric artery bridge was noted, and the initial set of baseline blood samples was obtained; thereafter, the bridge was placed in the pump head, and the pump speed was set to duplicate the preexisting flow rate. Once constant-flow perfusion was established, the second set of baseline blood samples was obtained, and then tamponade was induced as described previously. Repeat blood samples were obtained 5, 15, and 30 min after tamponade was established, and blood replacement was carried out in newborn subjects as previously described. Five subjects in each age group were studied in this protocol.

The use of controlled-flow perfusion may blunt the response of vasoconstrictors. This potential occurrence might attenuate the effects of extrinsic or systemic humoral agonists elaborated during tamponade and thus amplify the effects of local effector systems. To address this confounding variable, ANG II was infused in the femoral-mesenteric artery circuit (9 × 10-9 M/min) in a separate group of four subjects in the 3-day-old group, under both controlled-flow and autoperfused conditions. In this instance, autoperfused conditions were achieved by allowing blood to flow freely through the femoral-mesenteric artery circuit. Two subjects were administered ANG II under autoperfused and then controlled-flow conditions, whereas the order of perfusion was reversed in the other two subjects. In both instances, 1 h elapsed between ANG II infusions to minimize the effects of tachyphylaxis.

Protocol V: Effect of subpressor dose of ET-1 on the contractile response to ANG II in mesenteric artery rings. The goal of this protocol was to determine the effect of a subpressor dose of ET-1 on the contractile response to ANG II in mesenteric artery rings from 3- and 35-day-old subjects. The subpressor dose of ET-1 used in this protocol was 5 × 10-11 M, established in the first protocol as the highest concentration of ET-1 that did not cause a significant change in ring tension (Fig. 1). ANG II was administered in progressively increasing concentrations (10-11 to 10-7 M) in control rings and rings pretreated with ET. This protocol was carried out in paired rings from four subjects in each age group.


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Fig. 1.   Effects of constrictor agonists on mesenteric artery rings in vitro and intestinal vascular resistance in vivo. Top: effects of phenylephrine (PE), angiotensin II (ANG II), endothelin-1 (ET-1), and vasopressin (VP) on isometric tension in endothelium-intact mesenteric artery rings from 3-day-old (bullet ) and 35-day-old (open circle ) subjects. Data are means ± SD; n = 3 subjects for all observations. Brackets denote concentration. Bottom: effects of sustained intra-arterial infusion of these pressors on vascular resistance across autoperfused in vivo intestinal loops prepared in 3-day-old (bullet  and black-triangle) and 35-day-old (open circle  and triangle ) subjects. B, baseline value; M, maximal change; S, final steady-state level; black-triangle and triangle , subjects pretreated with the respective blocking agent before agonist infusion (see text for infusion rates and concentrations). Data are means ± SD; n = 3 subjects for all observations. a P < 0.01, maximal change (M) vs. baseline (B); b P < 0.01, steady state (S) vs. maximal change (M); c P < 0.01, 3-day-old vs. 35-day-old subjects.

Measurement of Plasma ANG II and ET-1 Levels

ANG II and ET-1 were determined at baseline and after 30 min of tamponade in control, denervated, and constant-flow protocols, i.e., in 15 subjects in each age group. These protocols were chosen because they did not involve administration of blocking agents, which might have affected release of systemic vasoconstrictor agonists. Whole blood (2 ml) was drawn from the arterial catheter in a chilled syringe and was transferred in an iced polypropylene tube containing 2 mg EDTA and 1,000 kallikrein inhibitory units of aprotinin. Tubes were centrifuged at 1,600 g for 15 min at 0°C, and the plasma was decanted. An equal volume of 1% trifluoroacetic acid (TFA) was added to the plasma, which was then spun at 10,000 g for 20 min at 4°C. During this spin, sep-columns filled with 200 mg C18 were washed with 1 ml of 60% acetonitrile in 1% TFA and then with 3 ml of 1% TFA three times. Acidified plasma was loaded on the column and was washed with 3 ml of 1% TFA two times. The peptides were then eluted from the column with 3 ml of 60% acetonitrile in 1% TFA. The eluant was evaporated to dryness with a centrifugal concentrator. ANG II and ET-1 radioimmunoassays were then carried out using a kit obtained from Peninsula Laboratories. The cross-reactivity of the supplied antibodies to ET-1 were stated as <2% for ET-2 and ET-3, whereas that for the antibodies to ANG II were stated as <1% for ANG I. These data were confirmed in our laboratory.

Statistical Analyses

Data from the ring studies were expressed as a percentage of contraction noted in response to 80 mM KCl-Krebs buffer, which was determined in each ring. Comparison of the ED50 and IC50 data for each agonist-antagonist pair was determined by a two-way ANOVA that utilized age group and drug concentration as main effects. As well, the maximal contractile response noted in response to each agonist was compared between age groups by t-test.

Blood flow and vascular resistance data from the in vivo gut loop studies were expressed as a function of tissue weight. Tissue O2 uptake was determined as the product of the arteriovenous O2 content difference [(a-v)O2] and blood flow. Hemodynamic and oxygenation data were analyzed by a three-way ANOVA that utilized age group (3 vs. 35 days old), condition (unblocked vs. blocked), and time (baseline vs. 5, 15, and 30 min of systemic hypotension) as main effects. If the F statistic for the entire ANOVA was significant (p<.01), subsequent analysis of the main effects was carried out using Tukey B-tests to determine if the magnitude of change of the respective variable over time was significantly different between age groups, or between conditions (i.e., blocked vs. unblocked) within each age group. If the main effects defining age group or condition were significant, then post hoc t-tests were carried out among time points within each age group to determine the specific sites of significance.


    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

The ED50 values for PE, ANG II, VP, and ET-1 observed within in vitro mesenteric artery rings were similar in the two age groups studied, as follows: PE, 8 × 10-8 M; ANG II, 9 × 10-9 M; VP, 2 × 10-10 M; and ET-1, 6 × 10-9 M. Also, the maximal vasoconstriction observed during intra-arterial infusion of these agonists in vivo was similar when these data were expressed as a percent increase from baseline (Fig. 1). Autoregulatory escape from the vasoconstriction induced by sustained intra-arterial infusion of ET-1 and VP was very limited in both age groups (Fig. 1). In contrast, escape from the contractile effects of PE and ANG II was nearly complete, but only in older subjects; thus, intestine from 3-day-old subjects demonstrated a negligible degree of autoregulatory escape from these agonists (Fig. 1). The IC50 values for the blocking agents determined in mesenteric artery rings in vitro were similar in both age groups, as follows: phentolamine, 5 × 10-7 M; losartan, 2 × 10-7 M; PIA, 1 × 10-8 M; and BQ-610, 5 × 10-7 M. Intra-arterial bolus administration of a dose one order of magnitude greater than the IC50 to in vivo intestine blocked >90% of the contractile response to the respective agonists (Fig. 1).

Subjects from both age groups tolerated 30 min of tamponade-induced hypotension well. Blood gas tensions remained stable throughout the hypotensive period (Table 1), and very little drift in mean arterial pressure was noted. The effects of systemic hypotension on intestinal hemodynamics and oxygenation were age dependent; thus, intestinal vascular resistance increased, whereas intestinal oxygenation decreased during hypotension to a significantly greater degree in 3- than in 35-day-old subjects (Table 2, Figs. 1 and 2). O2 delivery, calculated as the product of blood flow and arterial O2 content, decreased to a similar degree in both age groups during hypotension. In contrast, the increase in intracellular (a-v)O2 [(a-v)O2i] during tamponade was significantly greater in older (+90%) than in younger (+55%) subjects (Table 2). Therefore, the relative inability of 3-day-old intestine to augment O2 extraction during hypotension significantly contributed to the reduction of tissue oxygenation noted at this time. The baseline plasma concentration of ANG II was greater in younger subjects; however, the concentrations reached after 30 min of hypotension were similar in both age groups and were both significantly greater than the corresponding baseline value (Fig. 3). The plasma concentrations of ET-1 at baseline after 30 min of hypotension were both greater in 3- than in 35-day-old subjects (Fig. 3). However, the increase in plasma ET-1 concentration observed in the younger group at 30 min of hypotension was not significantly different from the baseline value.

                              
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Table 1.   Arterial and mesenteric venous blood gas and oxygen content values before and during tamponade


                              
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Table 2.   Effects of systemic hypotension on intestinal hemodynamics and oxygenation in 3- and 35-day-old swine



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Fig. 2.   Gut vascular resistance before and during tamponade. Data are shown for the following 3 protocols: control (bullet  and open circle ), losartan ( and ), and BQ-610 (black-triangle and triangle ) for 3-day-old (bullet , , and black-triangle) and 35-day-old (open circle , , and triangle ) age groups. Data are given as means ± SD; n = 5 subjects for all observations. F statistics for the 3-way ANOVA for these data were significant (P < 0.01). BI, baseline; BII, 5 min after injection of agonist in mesenteric artery. Subsequent post hoc tests: a P < 0.01 vs. BI; b P < 0.01, change in the variable over time different from 3-day-old group; c P < 0.01, change over time different, losartan treated vs. BQ-610 treated, within each age group.


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Fig. 3.   Plasma concentrations of ANG II and ET-1 at baseline and during tamponade. These data were collected in the 3 protocols that did not incorporate administration of blocking agents (i.e., control, controlled-flow perfusion, and denervation). Data collected in these individual protocols were not different, so that all data points were combined for this analysis and data presentation. Data are given as means ± SD; n = 15 subjects for all observations. Open bars, 3-day-old subjects; hatched bars, 35-day-old subjects. F statistics for 2-way ANOVA were significant for both data sets. Subsequent analysis of main effects (Tukey B-test) determined significant differences between groups (b) and within each group over time (a), all at P < 0.01.

Two of the four blocking agents exerted significant effects on intestinal hemodynamics during systemic hypotension. Losartan, which blocks ANG AT1 receptors, significantly attenuated the rise in gut vascular resistance in both age groups at 15 and 30 min into hypotension; indeed, losartan virtually eliminated the hypotension-induced rise in gut vascular resistance in older subjects (Table 3, Fig. 2). Pretreatment with losartan significantly attenuated the reduction of gut O2 uptake during systemic hypotension in younger subjects (Fig. 4). This improvement in tissue oxygenation was mediated at two levels: first, the hypotension-induced reduction of gut blood flow, and thus O2 delivery, was less in losartan-treated newborn subjects, whereas the increase in (a-v)O2i, which reflects the diffusive phase of O2 transport, was greater after AT1 receptor blockade. A similar beneficial effect was not as obvious in older subjects, but this was primarily consequent to the fact that gut O2 uptake was not compromised significantly during hypotension under control conditions in this age group. BQ-610, which blocks ET-1 ETA receptors, decreased the hypotension-induced increase in gut vascular resistance in younger subjects, but only at the very end of the hypotensive period (Table 4, Fig. 1). This agent did not cause any significant change in tissue oxygenation during systemic hypotension. In a separate group of three subjects in each age group, both losartan (2 × 10-6 M) and BQ-610 (5 × 10-6 M) were administered in the mesenteric artery before induction of systemic hypotension. The combined effect of both blocking agents was slightly but not significantly greater than that noted after losartan alone (Table 5). Phentolamine, which blocks alpha 1-receptors, and PIA, which blocks VP receptors, had no effect on intestinal hemodynamics or oxygenation during hypotension in either age group (Tables 6 and 7). Similarly, complete denervation of the isolated gut loop had no effect on the response to hypotension in either group (Table 8).

                              
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Table 3.   Effects of systemic hypotension on intestinal hemodynamics and oxygenation in 3- and 35-day-old swine, in the presence of losartan



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Fig. 4.   Gut O2 uptake before and during tamponade. Data are shown for the following 3 protocols: control (bullet  and open circle ), losartan ( and ), and BQ-610 (black-triangle and triangle ) for 3-day-old (bullet , , and black-triangle) and 35-day-old (open circle , , and triangle ) age groups. Data are given as means ± SD; n = 5 subjects for all observations. F statistics for 3-way ANOVA for these data were significant (P < 0.01). Subsequent post hoc tests: a P < 0.01 vs. BI; b P < 0.01, change in the variable over time different from 3-day-old group; c P < 0.01, change over time different, losartan treated vs. BQ-610 treated, within each age group.

                              
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Table 4.   Effects of systemic hypotension on intestinal hemodynamics and oxygenation in 3- and 35-day-old swine, in the presence of BQ-610


                              
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Table 5.   Effects of systemic hypotension on intestinal hemodynamics and oxygenation in 3- and 35-day-old swine, in the presence of losartan and BQ-610


                              
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Table 6.   Effects of systemic hypotension on intestinal hemodynamics and oxygenation in 3- and 35-day-old swine, in the presence of the vasopressin receptor antagonist, PIA


                              
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Table 7.   Effects of systemic hypotension on intestinal hemodynamics and oxygenation in 3- and 35-day-old swine, in the presence of phentolamine


                              
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Table 8.   Effects of systemic hypotension on intestinal hemodynamics and oxygenation in 3- and 35-day-old swine, following surgical denervation of the gut loop

Intestinal hemodynamics were significantly different in gut loops perfused under controlled-flow conditions during systemic hypotension when compared with controls (Tables 3 and 9, Fig. 5). The early rise in gut vascular resistance (i.e., at 5 min) noted under control conditions was absent in both age groups when gut flow was held constant. Intestinal vascular resistance increased at 15 and 30 min in both age groups; however, the magnitude of increase observed in younger subjects was significantly less than that noted in controls. The (a-v)O2i was significantly lower after 15 and 30 min of hypotension when compared with baseline, in both age groups, despite the fact that intestinal flow rate remained constant because of controlled-flow perfusion. This reduction caused intestinal O2 uptake to fall significantly in both groups at these time points.

                              
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Table 9.   Effects of systemic hypotension on intestinal hemodynamics and oxygenation in 3- and 35-day-old swine, in gut loops perfused under controlled-flow conditions



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Fig. 5.   Gut vascular resistance before and during tamponade. Data are shown for the following 2 protocols: control (bullet  and open circle ) and controlled flow (black-triangle and triangle ) for 3-day-old (bullet  and black-triangle) and 35-day-old (open circle  and triangle ) age groups. Data are given as means ± SD; n = 5 subjects for all observations. F statistics for 3-way ANOVA for these data were significant (P < 0.01). Subsequent post hoc tests: a P < 0.01 vs. BI; b P < 0.01, change in variable over time different from 3-day-old group; c P < 0.01, change over time different, losartan treated vs. BQ-610 treated, within each age group.

Controlled-flow perfusion did not blunt the effect of exogenous vasoconstrictors on the intestinal circulation; specifically, the mode of perfusion (autoperfusion vs. controlled-flow perfusion) did not alter the response to ANG II infusion. Intestinal vascular resistance rose from 0.67 ± 0.07 to 1.30 ± 0.08 mmHg · ml-1 · min · 100 g during the peak response to ANG II infusion under autoperfused conditions, whereas this variable changed from 0.72 ± 0.08 to 1.32 ± 0.07 mmHg · ml-1 · min · 100 g during the peak response to the peptide under controlled-flow conditions (mean ± SD, n = 4 subjects). Moreover, the steady-state changes in gut vascular resistance, i.e., after the completion of the escape process, were not dependent on the perfusion mode, and the order of perfusion proved inconsequential.

Administration of ET-1 at a subpressor concentration altered the dose-response relationship for ANG II (Fig. 6). Administration of 5 × 10-11 M ET-1 to the myograph well had no effect on mesenteric artery tone in either age group. However, this action caused a significant leftward shift in the dose-response curve for ANG II in rings from 3-day-old subjects (ED50 values: 6 × 10-9 M control vs. 3 × 10-10 M with ET-1 added).


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Fig. 6.   Effects of ET-1 on contractile response to ANG II in mesenteric artery rings. Subpressor dose of ET-1 was added to myograph well (5 × 10-11 M); thereafter, ANG II was administered in progressively greater concentrations. open circle , Rings administered ET-1; bullet , control rings. Top: 3-day-old subjects; bottom: 35-day-old subjects. Data are means ± SE. ED50 data: 3-day-old: control, 6 × 10-9 M; ET-1, 3 × 10-10 M (P < 0.01); 35-day-old: control, 6 × 10-9 M; ET-1, 3 × 10-9 M (not significant).


    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Two novel observations were made during these experiments. First, vasoconstriction within intestine from 3-day-old subjects during systemic hypotension was mediated by ANG II and ET-1, as well as by vascular regulatory mechanisms intrinsic to the intestine; in contrast, vasoconstriction within intestine from 35-day-old subjects was mediated almost entirely by ANG II. Second, systemic hypotension compromised tissue O2 uptake in younger but not older intestine. Each of these observations will be discussed in turn.

Effects of Systemic Hypotension on Postnatal Intestinal Vascular Resistance

A substantial portion of the vasoconstriction noted in 3-day-old intestine in response to systemic hypotension was mediated by vasoactive effector systems intrinsic to the gut vasculature. This was most evident in the controlled-flow experiments, which essentially eliminated a local vascular response to hypotension by maintaining flow and pressure within the gut loop steady during tamponade. Under these conditions, the magnitude of vasoconstriction in response to systemic hypotension was significantly reduced in younger intestine (i.e., +79 vs. +59% in autoperfused vs. controlled-flow gut loops). This difference was not consequent to a nonspecific impairment of vasoconstriction generated by the use of controlled-flow perfusion because gut loops perfused under controlled-flow conditions demonstrated a vasoconstrictive response to ANG II similar to that noted under autoperfused conditions. Instead, it appeared to be an age-specific response of the intestinal circulation to the perturbation of reduced perfusion pressure, a response that was consistent with previous reports from this laboratory (22, 24). Newborn intestine, i.e., swine intestine from subjects <= 3 days of age, consistently fails to demonstrate locally mediated vasodilation, or autoregulation, in response to reduced perfusion pressure. Rather, this circulation consistently responds to reduced perfusion pressure by significant vasoconstriction, even when O2 delivery-to-demand ratio is reduced (22, 24). Reduction of this ratio generally enhances the intensity of autoregulation (10, 12). In contrast to the newborn circumstance, intestine from 35-day-old subjects generally demonstrates a modest degree of vasodilation in response to perfusion pressure reduction, a response that is clearly enhanced during arterial hypoxemia, ischemia, or increased tissue O2 demand (22, 24). The mechanistic basis for the unique behavior of newborn intestine is presently unknown.

ANG II was an important mediator of gut vasoconstriction during systemic hypotension in both age groups, as evidenced by the reduction of this response in subjects pretreated with losartan and also by the marked increase in plasma ANG II levels observed during tamponade. This finding was anticipated, based on the work of Bulkley and colleagues who demonstrated ANG II-induced vasoconstriction of the intestinal (2), colonic (6), gastric (3), and hepatic (7) circulations during tamponade-induced hypotension in adult subjects. Systemic hypotension caused a greater degree of vasoconstriction in younger (+59%) than in older (+14%) intestine perfused under controlled-flow conditions. The nature of the controlled-flow preparation makes it likely that the bulk of this constriction was mediated by systemic humoral agonists, i.e., the preparation eliminated the local vascular response to reduced perfusion pressure by maintaining pressure and flow constant and also eliminated all extrinsic neural input in the gut circulation. The greater response in younger intestine might reflect, in part, the combined effects of ANG II and ET-1; this possibility is especially evident on comparison of Figs. 2 and 5. However, it is also possible that the constrictor response to ANG II was more sustained in younger intestine. This possibility is reinforced by review of Fig. 1, where it is clear that autoregulatory escape (16) from exogenously administered ANG II occurred in 35- but not in 3-day-old intestine. The time course of the tamponade experiments makes it most likely that the ANG II-mediated vasoconstriction noted during systemic hypotension reflected the escape phase rather than the peak response to ANG II. Lack of escape in younger intestine would thus result in sustained ANG II-induced contraction.

ET-1 participated in the vascular response to systemic hypotension in 3-day-old intestine but not 35-day-old intestine, and it was only noted at 30 min. ET-1 is a potent, long-lasting constrictor produced at several sites within the circulatory system, including cardiac myocytes and endothelial cells, and its release during systemic cardiovascular instability has been reported (26, 29). ET-1 and ANG II interact in several circulations, each amplifying the response of the other (14, 20, 31). It was in this context that protocol involving in vitro rings of mesenteric artery cut was carried out to determine if the effects of ANG II and ET-1 were synergistic within the 3-day-old intestine. The results were clearly mixed. Prior administration of a subpressor dose of ET-1 enhanced the contractile effect of ANG II within in vitro rings of mesenteric artery from 3- but not from 35-day-old subjects. However, the effects of combined administration of losartan and BQ-610 on hypotension-induced vasoconstriction in 3-day-old intestine in vivo were not significantly different from the effects of losartan alone. Stated otherwise, combined blockade of ANG II and ET-1 receptors did not have a significantly additive effect in vivo. One explanation for this discrepancy might be the relative ability to detect change within the in vitro and in vivo preparations; a modest increase in isometric tension might be observed in a system designed to quantify tension at the 0.1-g level, whereas the capacity to measure vascular resistance across the entire intestine is less sensitive. From a physiological standpoint, however, the in vivo observations are clearly more relevant. A greater role for ET-1 might have been noted if the time course of the in vivo work had been extended beyond 30 min, because regulation of ET-1 release occurs at the level of transcription-translation, which might take longer than 30 min to occur (4, 26).

Effects of Systemic Hypotension on Postnatal Intestinal Oxygenation

Intestinal O2 uptake was compromised during systemic hypotension to a significantly greater extent in 3- than in 35-day-old subjects. The difference was partly consequent to the greater reduction in intestinal blood flow noted in younger subjects during systemic hypotension. This action reduced the convective phase of O2 transport, or O2 delivery, calculated as the product of blood flow and arterial O2 content (10, 12). It is also likely that the perfused capillary density within the intestinal microvasculature also decreased in response to systemic hypotension, especially in younger subjects. The best evidence of this effect is the reduction of (a-v)O2 noted during hypotension in intestine perfused under controlled-flow conditions. In the presence of a constant flow rate, (a-v)O2 should change only if tissue O2 demand increases or if the perfused capillary density decreases (10, 12). This latter circumstance decreases the surface area available for O2 diffusion from capillary to cell, an effect that limits the diffusive phase of O2 transport and reduces O2 extraction (11). It is unlikely that intestinal O2 demand increased during systemic hypotension, especially in light of the fact that neither ANG II nor ET-1 directly alters intestinal metabolic rate (15). It is likely, however, that ANG II and/or ET-1 released in response to systemic hypotension constricted precapillary sphincters, microvascular elements that govern perfusion of individual capillaries within the intestinal microcirculation (10-12).

Based on these observations, it is possible to conclude that the significant vasoconstriction noted in 3-day-old or newborn intestine during systemic hypotension is mediated by ANG II and ET-1 and also by the local vascular response to reduced perfusion pressure. These effects compromise both the convective and diffusive phases of O2 transport and result in substantial compromise of intestinal oxygenation. Moreover, these effects are clearly age specific, as they occur to a greater extent in very young subjects. In 1969, Lloyd (17) postulated that significant intestinal ischemia occurred in human infants during asphyxia or hypotension secondary to activation of extrinsic constrictor systems, especially the sympathetic nervous system. This notion was verified later in a piglet model by Touloukian et al. (27) and Alward et al. (1), both of whom demonstrated marked reduction of intestinal perfusion when systemic cardiovascular stability was compromised. Data presented in this report indicate that ANG II and ET-1, rather than sympathetic efferents, cause vasoconstriction in newborn intestine during systemic hypotension. However, these observations clearly support Lloyd's original contention that the newborn intestine is uniquely vulnerable to systemic circulatory events.


    ACKNOWLEDGEMENTS

Chuck Miller and David Dunaway provided excellent technical support for this project.


    FOOTNOTES

This work was supported by National Institute of Child Health and Human Development Grant HD-25256.

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: P. T. Nowicki, Children's Hospital, 700 Children's Dr., Columbus, OH, 43205.

Received 13 July 1998; accepted in final form 14 October 1998.


    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Gastroint Liver Physiol 276(2):G341-G352
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society




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