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
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ABSTRACT |
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
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
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INTRODUCTION |
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
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.
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METHODS |
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
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 ( ) and 35-day-old ( ) 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 ( and ) and 35-day-old ( and )
subjects. B, baseline value; M, maximal change; S, final
steady-state level; and , 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.
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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.
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RESULTS |
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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Fig. 2.
Gut vascular resistance before and during tamponade. Data are shown for
the following 3 protocols: control ( and ), losartan ( and
), and BQ-610 ( and ) for 3-day-old ( , , and ) and
35-day-old ( , , and ) 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.
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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
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 ( and ), losartan ( and ), and BQ-610 ( and ) for
3-day-old ( , , and ) and 35-day-old ( , , and ) 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
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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 ( and ) and controlled flow
( and ) for 3-day-old ( and ) and 35-day-old ( and )
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.
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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. , Rings
administered ET-1; , 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).
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|
 |
DISCUSSION |
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.
 |
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