Departments of Pediatrics and Physiology, The Ohio State University and The Wexner Institute for Pediatric Research, Children's Hospital, Columbus, Ohio 43205
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ABSTRACT |
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This laboratory has previously reported that sustained reduction of blood flow in newborn intestine causes a triphasic increase in vascular resistance that occurs over 3-4 h and that these changes are mediated, in part, by loss of endothelial nitric oxide (NO) production. This study examines the effects of exposure to sustained low-flow perfusion on the subsequent response to three contractile agonists: ANG II, norepinephrine (NE), and endothelin-1 (ET-1). Gut loops from 3- and 35-day-old swine were exposed to low-flow conditions in vivo (i.e., reduction of flow to ~50% of baseline) for 30 min or 5 h. Thereafter, they were removed to an extracorporeal perfusion circuit for in vitro hemodynamic assessment; alternatively, the mesenteric artery perfusing the gut loop was removed and cut into rings for assessment of isometric tension development. Gut loops from 3-day-old subjects exposed to low-flow conditions demonstrated significantly increased contractile responses to ANG II, NE, and ET-1; also, mesenteric artery rings from these gut loops demonstrated a significant reduction of the ED50 for all three agonists. Similar changes were not observed in intestine or mesenteric artery rings from older subjects. Sustained blockade of endogenous NO synthesis with NG-monomethyl- L-arginine duplicated the effects of exposure to sustained low-flow perfusion. It appears that sustained reduction of blood flow in newborn intestine decreases constitutive NO production, which in turn causes a generalized enhancement of the contractile efficacy of ANG II, NE, and ET-1.
newborn; nitric oxide; angiotensin II; norepinephrine; endothelin-1; intestinal blood flow
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INTRODUCTION |
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THE NEWBORN INTESTINAL circulation differs from its adult counterpart in that its flow rate, when expressed as a function of tissue weight, is very high because its vascular resistance is very low (7, 19). This circumstance is mediated, in part, by nitric oxide (NO), as evidenced by the significant vasoconstriction that follows blockade of endogenous NO synthesis in vivo (17). These unique hemodynamic characteristics of newborn intestine may have significant physiological relevance, as they ensure adequate oxygen delivery to the intestine during the critical transition from fetal-to-newborn life when the gut assumes sole responsibility for nutrient assimilation. Also, the relative intestinal hyperemia during early newborn life provides abundant hepatic perfusion via the portal circulation, in essence duplicating fetal conditions wherein the umbilical vein had supplied the liver with substantial blood flow (22).
A vasculature designed to exist in a relatively high-flow state might be compromised when inflow pressure or flow rate falls. This predication has been proven correct for the newborn intestinal circulation. This circulation fails to autoregulate in response to reduced perfusion pressure (19). Moreover, mechanical reduction of flow rate to ~50% of baseline in an isolated gut loop within 3-day-old swine for a sustained period, i.e., 2-4 h, leads to progressive vasoconstriction (18). In contrast to the newborn, older swine who have completed the transition from a milk-based diet to cereal (i.e., weanlings, ~35 days old) demonstrate modest autoregulation and do not demonstrate significant vasoconstriction when confronted with sustained flow reduction (18). Also, these older subjects maintain a much lower rate of intestinal perfusion and have a higher resting vascular resistance when expressed as a function of tissue weight (7, 19).
The progressive vasoconstriction observed in 3-day-old intestine during sustained low-flow perfusion occurs in three phases: an abrupt 20% increase that occurs immediately after flow is reduced, a second 15% rise that begins ~5 min after flow reduction and that ends ~25 min later, and a third 5% increase that occurs slowly over the next 2-3 h. Some insight into the mechanistic basis for these changes can be derived from blockade studies. Thus blockade of endogenous NO synthesis before flow reduction significantly attenuated the second rise in resistance, whereas blockade of the angiotensin AT1 receptor decreased the third and final increase. These observations suggest that loss of NO production and increased ANG II production or receptor sensitivity contribute to the second and third phases of resistance increase, respectively. Factors other than NO and ANG II must also be involved in this process, however. For example, loss of NO-induced dilator tone would cause vasoconstriction if, and only if, an underlying constrictor tone was present, waiting to be unmasked. This constrictor tone cannot be produced by ANG II because blockade of AT1 receptors did not affect the second phase of resistance increase. Other constrictors, such as norepinephrine (NE) or endothelin-1 (ET-1), might be responsible.
The goal of this experiment was to determine the effects of exposure to sustained flow reduction on the subsequent contractile response to ANG II, NE, and ET-1 in the intestine of 3- and 35-day-old swine. To this end, isolated but autoperfused and innervated gut loops were prepared and exposed to low-flow conditions, i.e., flow reduction to ~50% of baseline that was maintained for 30 min or 5 h. Other subjects were exposed to a continuous infusion of the L-arginine analog NG-monomethyl-L-arginine (L-NMMA) for 5 h in lieu of flow reduction to allow comparison of the effects of endogenous NO synthesis blockade with those of sustained flow reduction. Thereafter, one of the following two experimental models was used: 1) removal of the gut loops to an in vitro blood-reservoir perfusion system followed by infusions of ANG II, NE, or ET-1; or 2) removal and mounting of the mesenteric artery from the gut loop for in vitro measurement of isometric tension development in response to the three agonists.
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METHODS |
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Care and Handling of Experimental Animals
Studies were conducted on 3-day-old (range 2-4 days) and 35-day-old (range 32-40 days) swine obtained from a local breeding farm on the day before study. Animals were kept in a large run dedicated to swine and were fed an age-appropriate diet until 12 h before surgery, when food, but not water, was withheld. Anesthesia was induced with xylazine (5 mg/kg im) and telezol (7.5 mg/kg im) and was maintained with pentobarbital sodium (5 mg/kg iv) given at 1-h intervals or sooner if deemed necessary by vivarium staff members. Animal care was provided in accordance with the Guide for the Care and Use of Laboratory Animals [Department of Health and Human Services (National Institutes of Health) Publication No. 85-23, Revised 1985], and all experimental protocols were approved by the Institutional Animal Care and Use Committee of the Children's Hospital Research Foundation.Creation of Intestinal Low-Flow Perfusion In Vivo
Rationale for preparation design. Exposure to low-flow conditions was carried out in vivo to allow changes in vascular reactivity to occur in a physiologically relevant setting, i.e., during autoperfusion with the subject's own, albeit heparinzed, blood. Intestinal hemodynamics were continuously monitored during this time to standardize the perturbation. Thereafter, one of two options was chosen. In some gut loops, an in vitro gut loop was created to allow administration of agonists in the absence of confounding systemic neurohumoral variables. In other gut loops, the mesenteric artery was harvested and prepared as rings for isometric tension recording.Initial preparation. Subjects were
anesthetized and ventilated to maintain normal blood gas tensions. A
femoral artery-vein pair was cannulated to allow measurement of
systemic arterial pressure, to obtain arterial blood samples for gas
analysis, and to allow infusion of crystalloid (5% dextrose in 0.9%
saline at 15 ml · kg1 · h
1),
additional anesthetic, and to return blood from the gut loop circuit. A
segment of distal jejunum-proximal ileum ~25 cm long was vascularly
isolated from the remainder of the gut so that it was perfused and
drained by a single artery-vein pair. The subject was administered
heparin (500 U/kg). The vein leading from the isolated gut loop was
cannulated, and this catheter was directed to a beaker primed with 50 ml of heparinized swine blood obtained from a previous study. Blood
collected in the beaker was returned to the animal at a rate equal to
venous outflow, thus maintaining euvolemia within the study subject. An
electromagnetic flowmeter (2.0 mm ID; Gould) and pressure transducer
were placed within the venous circuit to measure flow and pressure,
respectively. The position of the mesenteric vein catheter was adjusted
to keep venous pressure at 0 mmHg. The periarterial mesenteric nerves were carefully dissected off the mesenteric artery leading to the
isolated gut loop, which was then encircled by a silk thread. A fine
needle (27 gauge, 1.0 mm ID) was inserted in this artery ~1 cm
downstream from the site of the silk tie and was secured with a single
drop of glue. This intramesenteric artery catheter was connected to a
pressure transducer to monitor mesenteric artery pressure distal to the
induced coarctation so that an accurate assessment of gut vascular
resistance could be obtained. This catheter was also used to infuse
L-NMMA in some subjects during the in vivo portion of the
protocol, in place of flow reduction. The intestine and abdominal
incision were covered with plastic film, and temperature was kept at
38°C by means of servo-controlled heating elements placed above and
below the subject.
Creation of a low-flow state in the isolated gut
loop. The subject was left undisturbed for ~30 min or
until systemic pressure and gut blood flow achieved steady state,
defined as fluctuation of <5% over 10 min. A solid plastic cylinder
was placed between the silk tie and mesenteric artery, the tie was
tightened, and the cylinder was withdrawn. This action created a
coarctation of the vessel, the diameter of which approximated that of
the cylinder, and reduced the flow rate to the isolated gut loop. Different initial levels of flow reduction were used in each age group
because it has been previously shown that the effects of sustained
mechanical flow reduction on intestinal hemodynamics are age dependent:
resistance increases ~50% over 4 h in autoperfused, innervated gut
loops from 3-day-old subjects but only ~9% in 35-day-old subjects
(16). This difference would cause significantly lower flows in younger
subjects by the end of the protocol. Accordingly, mechanical flow
reduction was initially set to reduce flow to ~55% of baseline in
3-day-old subjects and ~45% of baseline in 35-day-old subjects.
During the course of the protocol, the greater vasoconstriction that
occurred in younger intestine in response to mechanical flow reduction
caused the final flow rates to be equally reduced in both age groups
with respect to their baseline values. The position of the mesenteric
vein catheter was adjusted to restore venous pressure to 0 mmHg 1 min
after flow reduction but was not adjusted again until the in vitro
portion of the protocol began. Arterial blood samples (0.3 ml) were
taken hourly to reassess blood gas tensions, and ventilator adjustments
were made when necessary to keep
PCO2,
PO2, and pH at their initial baseline
values. Control subjects were treated in an identical manner, except
that the mesenteric artery tie was not tightened. In other subjects,
L-NMMA was infused directly in the mesenteric artery
leading to the isolated gut loop for 5 h, at a rate of 0.3 ml/min
(104
M · kg
1 · h
1),
in lieu of mechanical flow reduction.
Experiment 1: Studies Using In Vitro Gut Loops
Preparation. At the end of the low-flow perfusion period, the artery leading to the gut loop was rapidly cannulated at the insertion site of the 27-gauge needle, the catheter was connected to an extracorporeal perfusion circuit, and the gut loop was severed from the study subjects and placed in a warmed (38°C) humidified plastic chamber. The arterial pressure was set to precisely duplicate the postcoarctation intestinal pressure noted just before arterial cannulation. It is important to note that flow was never restored to its baseline value, i.e., reperfusion was not permitted at any time during the protocol. The extracorporeal perfusion circuit consisted of a reservoir filled with heparinized, oxygenated blood obtained from a separate donor subject; the blood was continuously stirred and kept at 38°C. Flow to the gut loop was achieved by pressurizing the reservoir with 95% air-5% CO2 by means of a low-range air-pressure regulator that allowed precise control over arterial pressure (±1 mmHg); also, exposure to this gas maintained PO2 and PCO2 of the reservoir blood at 120 ± 5 and 37 ± 3 mmHg, respectively. This perfusion circuit provided pulseless perfusion with arterial pressure as the controlled variable. The vein circuit leading from the gut loop was diverted from the study subject to a waste beaker, and venous pressure was kept at 0 mmHg. In those subjects who had received a continuous infusion of L-NMMA during the in vivo portion of the experiment, the drug infusion was via a T-connecter within the arterial circuit at the identical drug infusion rate. Donor blood was obtained from ~90-day-old swine with an average weight of 40 kg and calculated blood volume of 4 liters (9). Donors were anesthetized, ventilated, and heparinized (500 U/kg), and 1 liter of blood was removed via a carotid artery catheter. During phlebotomy, 0.9% saline was infused in a jugular vein catheter at a rate equal to blood withdrawal to avert significant activation of the systemic renin-angiotensin axis. Arterial blood pressure within the donor subject remained stable during phlebotomy (89 ± 6 vs. 84 ± 5 mmHg before vs. immediately after phlebotomy). Blood was filtered two times (40 µm mesh) and was placed in the arterial reservoir. Each phlebotomy provided sufficient blood for two experiments, which were run in parallel using duplicate extracorporeal circuits.Protocol. The goal of this protocol
was to compare the effects of exposure to low-flow conditions or
L-NMMA on the subsequent response to a continuous infusion
of ANG I, ANG II, ET-1, or NE. The following four conditions were used
in this protocol: 1) control, i.e.,
infusion of an agonist in the mesenteric artery without prior
perturbation, 2) reduction of flow
rate in the isolated gut loop for 30 min in vivo, followed by infusion
of an agonist in the mesenteric artery after removal to in vitro
conditions, 3) reduction of flow
rate in the isolated gut loop for 5 h in vivo, followed by infusion of
an agonist in the mesenteric artery after removal to in vitro
conditions, or 4) infusion of
L-NMMA (104
M · kg
1 · h
1
at 0.3 ml/min) for 5 h in the mesenteric artery. All agonists were
infused at a rate of 0.3 ml/min until steady-state conditions were
attained; the drug infusion rates were
10
8 M/min for ANG II,
10
9 M/min for ET-1, and
10
8 M/min for NE. These
drug infusion rates were designed to provide an
ED50 of each agonist at the onset
of drug infusion. During agonist infusion, the arterial and venous
pressures across the gut loop were kept constant, i.e., pressure
adjustments were not carried out to compensate for the change in flow
rate caused by the contractile effect of the peptide. To confirm the
adequacy of NO synthesis blockade by L-NMMA, the response
to a bolus infusion of the NO-dependent dilator substance P was noted
after blockade was established in a separate group of animals. Infusion
of vehicle (0.9% saline) had no effect on the measured variables in
either age group.
Experiment 2: Studies Using Mesenteric Artery Rings
Preparation. At the completion of in vivo low-flow perfusion, the gut loop was removed from the study subject to a tray filled with iced Krebs buffer. The mesenteric artery within this tissue was removed, cleaned of adherent tissue, and cut into 3-mm rings with care taken to avoid contact with the intimal surface. Rings were mounted between two wire stirrups placed within a water-jacketed myograph to allow measurement of isometric tension. The myograph well was filled with Krebs buffer (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, aerated with 95% O2-5% CO2, pH 7.4). Rings were stretched to the optimal point on their length-tension curves, determined by noting the contractile response to 80 mM KCl-Krebs buffer. Endothelial integrity was tested in all rings by noting dilation ofProtocol. The goal of this protocol
was to determine if the enhanced response to contractile agonists was
accompanied by a lower ED50, i.e.,
the agent concentration necessary to cause 50% of the maximal
response. This protocol was only carried out on mesenteric artery rings
removed from 3-day-old subjects, and only two conditions were applied:
control and exposure to low-flow conditions for 5 h in vivo. All rings
had an intact and functional endothelium, and all constrictor agonists
were applied while the rings were quiescent, i.e., stretched to the
optimal point of their length-tension curve, but not precontracted.
Rings were administered progressively increasing concentrations of
contractile agonists in a stepwise manner. The dose ranges were as
follows: ANG II, 1011 to
10
7 M; ET-1,
10
11 to
10
6 M; and NE,
10
10 to
10
5 M. Each dose was given
only after steady-state tension had been reached after the preceding
dose. Each ring was administered only one agonist, and all observations
were made in paired rings.
Statistical Analysis
Gut loop data were analyzed by a three-way ANOVA that utilized age group (3 vs. 35 day old), condition (control vs. low-flow exposure vs. L-NMMA exposure in vivo), and time (baseline vs. peak contraction vs. steady-state contraction) as main effects. If the F-statistic was significant (P < 0.01), subsequent two-way ANOVAs were carried out to determine if the magnitude of change over time was different between specific conditions within each group or between groups. The effects of ANG II, ANG I, NE, and ET-1 on vascular resistance were also evaluated by expressing values noted after drug administration as a function of baseline resistance. This data transformation was carried out to clarify differences between age groups, inasmuch as baseline vascular resistance is substantially different in 3- and 35-day-old intestine. Data from mesenteric artery ring studies were expressed as a percentage of the maximal contraction noted in response to 80 mM KCl-Krebs buffer and were used to calculate the ED50, i.e., the agonist concentration that caused 50% of the maximum contraction. The ED50 and maximum contraction noted for each agonist were compared by a two-way ANOVA that utilized age group and condition (control vs. exposure to low-flow conditions) as main effects. ![]() |
RESULTS |
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Creation of Low-Flow Perfusion Within In Vivo Gut Loops
Mechanical reduction of intestinal flow rate caused a progressive vasoconstriction in both age groups that reached a plateau between 3 and 4 h after constriction of the mesenteric artery (Table 1). The response was significantly greater in 3-day-old subjects, both when low flow was sustained for 30 min (percent increase in resistance: 22% in 3-day-old subjects and 4% in 35-day-old subjects) and for 5 h (percent increase in resistance: 44% in 3-day-old subjects and 7% in 35-day-old subjects). However, because the initial degree of mechanical flow reduction was greater in older subjects, both groups demonstrated flow rates ~38% of their respective baseline values at the completion of the 5-h period. Actually, the 35-day-old group was exposed to a greater degree of flow reduction for a longer period of time, inasmuch as the flow reduction in older subjects was primarily achieved by mechanical means at the outset, whereas that in younger subjects developed slowly as vasoconstriction developed. Sustained infusion of L-NMMA increased vascular resistance in both age groups, but the effect was greater in younger subjects (Table 2). The rate of L-NMMA infusion was sufficient to block endogenous NO production, as evidenced by loss of a vasodilator response to the NO-dependent vasodilator agent substance P, given directly in the mesenteric artery (data not shown). Finally, conversion from in vivo to in vitro perfusion did not significantly affect hemodynamics in either age group (data not shown).
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Gut Loop Studies
The effects of exposure to low-flow perfusion were clearly age dependent (Fig. 1 and Tables 3-5). All three constrictor agents, i.e., ANG II, NE, and ET-1, caused a significantly greater peak level and steady-state vasoconstriction in gut loops exposed to low-flow perfusion in 3- but not 35-day-old subjects. The effect on ANG II- and ET-1-induced vasoconstriction was present after a 30-min exposure to low-flow conditions, whereas the effect on NE-induced vasoconstriction was only noted after 5 h of low-flow perfusion. Another age-specific difference observed during infusion of constrictor agents was the presence of escape, i.e., vasodilation that occurs despite continued application of the vasoconstrictor agonist. Gut loops from older subjects demonstrated escape from ANG II- and NE- but not from ET-1-induced contraction; in contrast, intestine from 3-day-old subjects only demonstrated escape from NE-induced vasoconstriction. In 3-day-old subjects, sustained in vivo infusion of L-NMMA caused changes in the response to ANG II, NE, and ET-1 similar to that noted after exposure to 5 h of low-flow perfusion. L-NMMA infusion had little effect in older subjects, although it did eliminate the escape from ANG II-induced vasoconstriction.
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Infusion of ANG I increased gut vascular resistance to a similar extent
in both age groups (Fig. 2 and Table
6). This contractile effect was virtually
eliminated in gut loops pretreated with captopril, indicating that the
vascular effect was mediated by local conversion of ANG I to the active
form of the peptide, ANG II (data not shown). Older subjects
demonstrated a significant escape from the ANG I-induced contraction,
whereas younger subjects did not. Exposure to low-flow conditions for
30 min had no effect on the response to ANG I in either age group
(Table 6 and Fig. 2). However, exposure to these conditions for 5 h
significantly increased the peak contraction noted during ANG I
infusion in both age groups, although escape from the ANG II-induced
contraction was still present in older subjects. Pretreatment of the
gut loops exposed to 5 h of low-flow perfusion with captopril virtually
eliminated the contractile response to ANG I.
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Vascular Ring Studies
Ring studies were only carried out in 3-day-old subjects, inasmuch as older subjects failed to demonstrate significant change in gut loop studies. In all experimental subjects, gut loops were exposed to 5 h of low-flow perfusion in vivo before vessel harvest and mounting. A significant leftward shift in the dose-response curve was observed for all three constrictor agents (Fig. 3). The calculated ED50 values were significantly lower in rings exposed to 5 h of low-flow conditions in vivo. Furthermore, the maximal degree of contraction noted in response to ANG II and ET-1 was significantly greater after exposure to low-flow perfusion, when expressed as a percentage of the contraction induced by 80 mM KCl-Krebs buffer.
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DISCUSSION |
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Effect of Exposure to Low Flow on the Subsequent Response to Contractile Agonists
The principal new finding from these experiments is that exposure of the intestinal circulation from 3- but not from 35-day-old swine to low-flow conditions causes a generalized increase in the response to constrictor agonists. The duration of exposure necessary to induce this effect was relatively brief, i.e., 30 min for ET-1 and ANG II, and the degree of flow reduction necessary to induce this effect, i.e., to 50% of baseline, was relatively moderate. It is thus unlikely that these changes were consequent to tissue hypoxia. Administration of L-NMMA duplicated the effects of exposure to sustained low-flow perfusion. This observation suggests that attenuation of NO production is responsible for the enhanced contractile effects of ANG II, NE, and ET-1, a conclusion that may also explain the age-specific nature of this effect. Constitutive production of NO is greater in 3- than in 35-day-old swine intestine, and the mechanostimulus of flow is an important determinant of this process (5, 8, 17, 20). Hence, loss of constitutitve NO production could occur in younger but not older subjects simply because it is present at a greater rate at the outset. Increased endothelial production of NO in response to increased flow rate or wall shear stress has been well documented (5, 24). To the best of my knowledge, the inverse response, i.e., attenuation of the constitutive production of NO after flow reduction, has not been reported. On the basis of the current understanding of the means by which endothelial NO synthase activity is regulated (15), however, it seems feasible to expect that a reduction in flow rate across the endothelium could diminish the stimulus to endothelial NO synthase, leading to decreased NO production. Thus the activity of endothelial NO synthase is contingent upon the concentration of Ca2+-calmodulin within the cytosol (15), and mechanotransducers exert their effect on this enzyme by altering intracellular Ca2+ concentration ([Ca2+]i; see Ref. 8). Increased flow rate raises [Ca2+]i (27). It seems logical to expect that reduced flow rate has the inverse effect; indeed, anything that would reduce [Ca2+]i should decrease NO production.How might a reduction of NO alter the contractile response to ANG II, NE, and ET-1? There are at least three possibilities. First, relative removal of NO from the microvascular environment would alter the balance between constrictor and dilator stimuli, favoring constriction; indeed, several laboratories have reported enhanced contractile responses after ablation of endogenous NO synthesis (11, 21, 29, 33). Second, NO might affect the binding affinities of the ANG II, NE, and ET-1 receptors, specifically by altering the coupling of these receptors to their respective G proteins. In this context, Miyamoto et al. (16) reported significant attenuation in the affinity of the bradykinin BK2 receptor by exogenous NO. The presence of NO uncouples receptors from their G proteins, an action that reduces the receptor affinity for its ligand. The reverse circumstance might explain the present findings. Thus reduction of NO within the microvascular environment would increase receptor-G protein coupling, enhancing receptor affinity. Data from the ring studies provide indirect support for this notion: the ED50 values for ANG II, NE, and ET-1 were significantly lower in mesenteric artery exposed to sustained low-flow perfusion in vivo. Third, NO might affect expression of the receptors for ANG II, NE, and ET-1. In this context, downregulation of the AT1 receptor by NO has been reported (4, 11). Once again, the reverse circumstance, i.e., reduced NO prevalence resulting in enhanced receptor expression, might have occurred. This notion seems particularly feasible inasmuch as NO is most likely acting to inhibit the promoters that regulate receptor expression. Clearly, these explanations are speculative in nature.
Effects of Flow Rate on the Response to ANG I
ANG I, the precursor to ANG II, is not vasoactive; vasoconstriction after application of ANG I requires its conversion to ANG II by angiotensin-converting enzyme (ACE). This conversion clearly occurred within intestine from 3- and 35-day-old subjects, as evidenced by contraction in response to ANG I, as well as by elimination of the response after pretreatment with the ACE inhibitor captopril. Exposure to 30 min of low-flow perfusion had no effect on the response to ANG I in either age group. More sustained exposure, however, increased the contractile response to ANG I in all subjects. One interpretation of these observations is that ACE activity increased during sustained low-flow perfusion. Shear stress-responsive elements have been identified within the ACE promoter (30). Additionally, Rieder et al. (25) reported suppression of ACE expression in response to an increase in shear stress. It seems reasonable to speculate that the opposite effect might also occur, i.e., that ACE expression would increase if the suppressing stimulus of flow was reduced. Another possibility is that reduction of flow rate increased expression of endothelin (14), as this peptide has been identified as a stimulus to ACE expression in vascular smooth muscle (13). The time frame of this experiment, i.e., 5 h, is well within that necessary to engage transcriptional and translational machinery to increase ACE.Escape From ANG II, NE, and ET-1-Induced Contractions
"Escape" from sustained constrictor stimuli was first described in the gut circulation by Folkow et al. (9), who observed relaxation after peak contraction to sustained mesenteric nerve stimulation. Several explanations of the escape phenomenon have been offered; most have suggested that parenchymal-derived vasodilators produced in response to tissue hypoxia override the constrictor stimulus (28). In the present work, escape was both age and agonist specific; specifically, both age groups demonstrated escape from NE-induced contraction, whereas only older subjects demonstrated release from ANG II. This pattern suggests that the mechanisms responsible for the escape process are unique. Escape from the contractile effects of NE is reminiscent of that previously reported for sustained mesenteric nerve stimulation in postnatal swine intestine (3, 6). Escape from the contractile effects of ANG II was significantly attenuated by blockade of endogenous NO synthesis with L-NMMA, suggesting that NO participated in this response. AT1 receptors have been localized to endothelial and vascular smooth muscle cells of bovine and rat carotid artery (23, 32). Stimulation of the muscle receptor induces vasoconstriction, whereas ligand binding to the endothelial receptor causes increased NO production that offsets the constriction to some extent (2, 26). A similar process could explain escape from ANG II-induced contraction in older subjects; thus, the initial contraction is later attenuated by endothelium-derived NO production. If this explanation is correct, then one might surmise that endothelial AT1 receptors are developmentally regulated in the postnatal intestine, becoming functional several weeks after birth.Role of Constrictors in the Response of Newborn Intestine to Sustained Low-Flow Perfusion
Intestine from 3-day-old subjects demonstrates a triphasic vasoconstriction when its flow rate is mechanically reduced to ~50% of baseline (18). The second phase of this response appears to be consequent to a loss of NO production in response to flow reduction. Thus blockade of endogenous NO synthesis significantly increases baseline vascular resistance in 3-day-old intestine and virtually eliminates the second phase of resistance increase in response to mechanical flow reduction. The vascular resistance attained after L-NMMA infusion is very similar to that observed 30 min after mechanical flow reduction in newborn intestine (0.90 ± 0.04 vs. 0.92 ± 0.04 mmHg · ml ![]() |
ACKNOWLEDGEMENTS |
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David Dunaway and Chuck Miller provided outstanding technical assistance in this project.
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FOOTNOTES |
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This work was supported by National Institute for 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 and other correspondence: P. T. Nowicki, Children's Hospital, 700 Children's Dr., Columbus, OH 43205 (E-mail: nowickip{at}pediatrics.ohio-state.edu).
Received 23 September 1998; accepted in final form 12 February 1999.
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