From the Divisions of Neonatology and Molecular Medicine, Department of Pediatrics, Ohio State University and the Children's Research Institute, Children's Hospital, Columbus, Ohio 43205
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ABSTRACT |
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Developmental changes in the expression of endothelial nitric oxide synthase (eNOS) within the mesenteric artery of swine were studied in fetal (110 days postconception/117 days total gestation) and on postnatal days 1, 3, 10, and 30. Subjects in the 1-day-old group were subdivided into fed and nonfed. Transcription of eNOS was determined by real-time PCR, protein expression was evaluated by Western blotting, and hemodynamic and oxygenation parameters were measured within in situ gut loops before and after the administration of NG-monomethyl-L-arginine (L-NMMA). The abundance of eNOS mRNA remained steady throughout all ages. In contrast, expression of eNOS protein was twofold greater in the 1-day-old fed subjects compared with fetal or 1-day-old nonfed subjects. eNOS protein expression remained elevated on day 3, increased on day 10, and then declined to a level similar to the day 1 nonfed group by postnatal day 30. Intestinal vascular resistance was 31% lower in the day 1 fed group when compared to the day 1 nonfed group; resistance continued to decline through day 10 but then significantly increased on day 30. We conclude that the expression of eNOS changes within the mesenteric artery during early postnatal development at a posttranscriptional level.
newborn intestine; necrotizing enterocolitis
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INTRODUCTION |
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THE ROLE OF NITRIC OXIDE (NO) in regulation of porcine postnatal intestinal circulation is age specific, being more substantial in younger than older subjects (20, 23, 30). Thus blockade of endogenous NO synthesis with an L-arginine analog increases intestinal vascular resistance 45 ± 4% in 3-day-old piglets but only 8 ± 2% in 35-day-old subjects, whereas application of NO-dependent agonists (e.g., acetylcholine) causes a greater relaxation of phenylephrine-precontracted mesenteric artery rings from 3- than from 35-day-old piglets (20). Generation of cGMP by in vitro mesenteric artery under basal or stimulated conditions is greater in rings from 3- than from 35-day-old subjects (23). Finally, the concentration of total nitrates (NOx) is significantly greater within the effluent of buffer-perfused mesenteric arterial arcades from 3- than from 35-day-old piglets, and a direct relationship between the buffer flow rate, mesenteric arcade vascular resistance, and the concentration of NOx in the effluent is present only in younger subjects (30). This relationship is consistent with the observation that flow-mediated dilation, a process that can be NO-dependent (17), can be elicited in the intestinal circulation of 3- but not 35-day-old piglets (24). The age-specific participation of NO in regulation of the intestinal circulation most likely contributes to creating the significantly lower vascular resistance across intestine from 3- than from 35-day-old subjects (25).
The mechanistic basis for age-specific participation of NO in regulation of the postnatal intestinal circulation has not been established, although two possibilities seem feasible. First, expression of the endothelial isoform of NO synthase (eNOS) could be developmentally regulated within the intestinal vascular endothelium, being substantially greater at the time of birth and receding as postnatal age increases. In this context, there is precedent for developmental regulation of eNOS during perinatal life; for example, North et al. (22) demonstrated upregulation of eNOS in the murine pulmonary circulation just before parturition, when the rate of pulmonary blood flow dramatically increases to accommodate postnatal lung function (27). The second possibility is that the environment in which eNOS exists in the newborn intestinal vascular endothelial cell might favor increased enzyme activity. Thus, although eNOS is considered a constitutive enzyme, its activity can be increased by the presence of Ca/calmodulin (18) or by phosphorylation of specific serine residues (13).
The goal of this work was to assess the first possibility, i.e., to determine whether eNOS expression changes during early postnatal life. To this end, real-time PCR was used to quantify eNOS mRNA expression and Western blotting was used to quantify eNOS protein expression in homogenates of mesenteric artery obtained from late gestation fetal subjects and subjects on postnatal days 1, 3, 10, and 30. These postnatal age groups were selected to reflect the dietary transition from sow's milk to grain that occurs during the first postnatal month in swine. Furthermore, subjects studied on postnatal day 1 were subdivided into two groups (nonfed and fed) to determine whether the onset of enteral feeding affected eNOS expression. In separate groups of 1-, 3-, 10-, and 30-day-old subjects, hemodynamic and oxygenation variables were measured within autoperfused, innervated, in situ gut loops under baseline conditions and after blockade of endogenous NO synthesis with the L-arginine analog NG-monomethyl-L-arginine (L-NMMA).
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MATERIALS AND METHODS |
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Age Groups and Animal Care
Choice of age groups. The age groups (postconceptual day 110; postnatal days 1, 3, 10, and 30) were selected to include landmarks in dietary change and thus intestinal workload during perinatal life that occurs in swine reared in their natural environment. The fetal intestine is functionally dormant in contrast to the active state initiated by the onset of postnatal feeding. Sow's milk has an exceptionally high fat content in the first postnatal week, during which time piglets feed so frequently that their stomachs are always full and gastric emptying is nearly continuous. Weaning to a cereal diet begins at the end of the first postnatal week, so that by postnatal day 10 the diet is a mixture of cereal mash and sow's milk. Complete removal of the piglets from the sow occurs early during the third postnatal week. By postnatal day 30, the piglet's diet has been entirely grain-based for over a week and the frequency of feeding has decreased from 10-12 times per day to twice a day (2, 12, 33). Piglets of both sexes were studied in all age groups.
Acquisition of fetal subjects. A time-dated pregnant sow was obtained from the swine breeder on the day of surgery. Delivery of the fetal subjects was achieved by cesarean section on postconceptual day 110 (0.95 gestation of 117-day gestational period). Sows were initially anesthetized with telezol (6 mg/kg im) and xylazine (7.5 mg/kg im) and maintained with 1% halothane. First the right and then the left uterine horn were entered, and three fetuses were removed from each sow in 15-min intervals, providing sufficient time for tissue removal from the delivered fetus before removal of subsequent subjects. Fetal subjects were used solely for harvest of mesenteric artery for real-time PCR and Western blotting for eNOS protein.
Acquisition of postnatal subjects: postnatal day 1. Subjects in the 1-day-old group were subdivided into nonfed and fed groups. This subdivision was accomplished by having the swine breeder remove the first three piglets in designated litters from the sow immediately after birth. These subjects comprised the 1-day-old nonfed group. They were placed into an incubator provided to the swine farm by the laboratory and were kept therein for 18 h after birth, during which time they were not allowed anything by mouth. Remaining piglets in these litters were allowed to suckle ad libitum for the same 18-h period. These piglets comprised the 1-day-old fed group, and four litters were studied in this manner.
Acquisition of postnatal subjects: postnatal days 3, 10, and 30. Subjects in the 3-, 10-, and 30-day-old groups were kept on the farm until the day of use, and all subjects in these groups were studied on precisely that postnatal day, i.e., day 3, 10, or 30; unless stated otherwise, age ranges were not used. All subjects in these age groups were allowed access to the age-appropriate diets as previously described.
Protocol 1: Studies of eNOS Expression
Tissue harvest. Fetal and postnatal subjects were anesthetized with telezol (6 mg/kg im) and xylazine (7.5 mg/kg im) followed by pentobarbital sodium (5 mg/kg iv). The distal portion of the mesenteric artery trunk and its ileal branches were removed for eNOS expression analysis. This portion of the mesenteric arterial arcade corresponded to that which would be present within the isolated gut loop (described below). One-half of these pieces were immersed in an RNase tissue inhibitor (RNAlater, Ambion, Austin, TX), whereas the other half were immersed into a homogenization buffer described below. Subjects in both subgroups of the postnatal day 1 age group were treated differently; thus they had a blood sample taken from the abdominal aorta for glucose, sodium, and blood gas analysis before aortic transection to provide an assessment of their metabolic status. Also, particular care was taken to determine the animal's state of hydration and gastrointestinal contents.
Real-time PCR methodology.
We chose to quantify eNOS mRNA using real-time PCR methodology inasmuch
as this approach provides the most precise means of mRNA
quantification, compared with conventional Northern blotting or RNA
protection assays. Total RNA was isolated from small (<0.3 mm) pieces
of whole mesenteric artery (i.e., adventita, muscularis, endothelial
layers) using a phenol-free total RNA isolation kit (RNAqueous,
Ambion). Isolated RNA was treated with RNase-free DNase to eliminate
DNA contamination, and quantity and quality of RNA was quantified by
spectrophotometry and electrophoresis on 1% formaldehyde denaturing
gels. Real-time PCR was carried out by a one-step method using an ABI
Prism 7700 sequence detection system (Applied Biosystems, Foster City,
CA). For amplification of the eNOS product, 100 ng total RNA isolated
from the mesenteric artery was added to a reaction mixture consisting
of Taqman buffer A consisting of (in mM) 5.5 MgCl2, 0.3 dATP, 0.3 dGTP, 0.3 dCTP, and 0.6 dUTP, with
0.025 U/µl AmpliTaqGold DNA polymerase, 0.25 U/µl multiscribe
reverse transcriptase, 0.4 U/µl RNase inhibitor, 300 nM forward and
reverse primers, and 150 nM probe in a final volume of 25 µl. The
complete cDNA for swine eNOS has been established (36),
and this sequence was used to generate the primers and a
fluorescence-tagged probe for the real-time PCR reaction. The real-time
PCR primers were nested within a second set of PCR primers generated
for swine eNOS that flank a 472-bp region from bp 1052 to bp 1533. This
second set of primers were used to amplify this segment of swine eNOS,
which was then sequenced to confirm validity. The primers and probe
used in the real-time PCR reaction flanked a 70-bp fragment from bp
1247 to bp 1317, as follows: forward primer 5'-TGGCCAAAGTGACCATTGTG-3';
reverse primer 5'-AAGCACCTGGAGAACGAGCA-3', probe
5'-TCACGCCGCCACGCCT-3'. The initial reverse transcription cycle was
carried out at 48°C for 30 min, followed by 2 min at 50°C. PCR
amplification was preceded by an incubation at 95°C for 10 min;
thereafter, a 40-cycle PCR was carried out consisting of 94°C
denaturation for 30 s, 58°C annealing for 1 min, and 68°C elongation for 1 min. A reaction mixture with ribosomal RNA primers and
probe was run with each set of mesenteric artery samples to control for
RNA loading. Ribosomal RNA has been used as a loading standard in other
studies dealing with the developmental expression of various proteins
(10); as well, preliminary work in our laboratory indicated stability of the rRNA signal within mesenteric artery during
the perinatal time interval studied. ABI Prism 7700 provides an
amplification curve constructed by relating the fluorescence signal
intensity (Rn) to the PCR cycle number. The threshold cycle value
(Ct) is the cycle at which an increase in
Rn above background occurs and is thus a direct marker of the abundance of the
mRNA in question, i.e., a low Ct indicates a substantial abundance of the mRNA and vice versa. Ct values obtained
for each mesenteric artery sample were plotted to the standard curve
prepared on the same day to determine the copy number of eNOS mRNA per nanogram of total RNA.
Generation of standard curve for eNOS mRNA.
A standard curve was generated for each PCR analysis to facilitate the
conversion of Ct values for each mesenteric artery sample
to an eNOS mRNA copy number, expressed per nanogram of total RNA. Swine
eNOS cDNA (36) was cloned into the pCMV-Script mammalian
expression vector (Stratagene, La Jolla, CA) to be used as a template
for in vitro transcription of swine eNOS mRNA. RNA was transcribed from
the template using T7 RNA polymerase and the Maxiscript Transcription
Kit (Ambion). Transcribed RNA was precipitated (LiCl/ethanol) and
purified, the RNA was quantified by spectrophotometry and sequence
analysis, and the copy number of eNOS mRNA was thus established.
Standards containing 104-109 copies of
eNOS mRNA were prepared, and real-time PCR was carried out by using
these standards as templates using conditions identical to those used
for the mesenteric artery samples. These standards consistently
provided a linear standard curve. A representative standard curve is
shown in Fig. 1.
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Western blotting.
The mesenteric artery and its primary branches were removed and
immediately immersed in liquid N2. Frozen tissue was
pulverized to a fine powder in a mortar filled with liquid
N2 and then suspended in homogenization buffer of the
following composition: 20 mM MOPS, 1% Triton, 4% SDS, 10% glycerol,
5.5 mM leupeptin, 5.5 mM pepstatin, 200 kallikrein inhibitory units
aprotinin, 1 mM Na3VO4, 10 mM NaF, 100 µM ZnCl2, 20 mM -glycerophosphate, and 20 µM
phenylmethylsulfonyl fluoride. Tubes were vortexed for 10 min and
centrifuged, and then the supernatant was recovered. Protein separation
was carried out by electrophoresis in 10% SDS-polyacrylamide gels (20 µg protein/lane) and then transferred overnight to polyvinylidene
difluoride membranes. After being blocked for 2 h in 10%
dried milk in PBS-Tween buffer, the membranes were incubated with a
commercially available primary antibody directed against eNOS (1:500;
BD Transduction Laboratories, Los Angeles, CA). Thereafter, membranes
were rinsed, incubated with horseradish peroxidase-conjugated secondary
antibody for 1 h (Calbiochem, La Jolla, CA), and then developed
using enhanced chemiluminescence (Pierce, Rockford, IL). Protein
isolated from human umbilical vein endothelial cells was used as a
positive control. All samples from each age group were run
simultaneously on a single gel, and each blot was stripped and reprobed
with
-actin to correct for sample loading. Preliminary studies
revealed that
-actin expression within mesenteric artery does not
change over the age range used in this study, thus validating its use as an internal loading standard for eNOS. Densitometry was carried out,
and the final value for eNOS was expressed as the quotient of the
eNOS/
-actin signals.
Protocol 2: Hemodynamic Studies
Preparation.
The distal-most portion of the mesenteric vein was cannulated;
thereafter, the ileum and colon were divided at the ileocecal valve and the terminal portion of the small intestine was
isolated from the remainder of the intestine with care taken to avoid
trauma to lymphatic and neural tissue. The result was an innervated, autoperfused gut loop ~25 cm in length (<25% of the entire small intestine) consisting of distal jejunum and proximal ileum perfused and
drained by the distal portions of the mesenteric artery trunk and
mesenteric vein. Mesenteric venous cannulation was preceded by the
administration of heparin (500 U/kg), and the catheter was directed to
a beaker primed with 25 ml of 0.9% saline. Blood was returned to the
animal via the jugular vein by a pump set at the rate of venous
outflow. An electromagnetic flowmeter (2.0 mm ID; Gould, Cleveland, OH)
and a pressure transducer were placed within the venous circuit to
measure flow rate and mesenteric venous pressure. A catheter was placed
in a jejunal branch of the mesenteric artery and advanced into the
mesenteric artery trunk upstream from the intestinal loop for drug
infusion. The preparation was maintained at 37°C by means of a
servo-controlled heating lamp and by covering the tissue and incision
with plastic wrap. Because of the vascular isolation created for the
gut loop, measurement of the rate of venous outflow is equal to
arterial inflow, which can be expressed as milliliters per minute or
corrected for tissue weight. Once the gut loop was created, 15 min were allowed to elapse, during which time steady-state hemodynamic conditions were established, as evidenced by the stability of blood
flow within 5% of baseline. The flow rate was recorded at this point,
while the gut loop still contained abundant chyme; thereafter, chyme
was removed from the isolated gut loop by a gentle stream of warm
(37°C) saline followed by air until the luminal effluent was clear
and sufficient time was allowed for new steady-state conditions to
emerge. During this time, which was generally 45 min, intestinal blood
flow consistently decreased 25% in the 1-day-old fed subgroup
and in the 3- and 10-day-old groups. Chyme was not present in the
intestinal lumen of the 1-day-old nonfed subgroup. To be consistent,
the same cleansing routine was applied to this subgroup; however, no
change in blood flow was noted in any subject in this subgroup after cleansing.
Experimental protocol.
Baseline hemodynamic measurements were obtained after the gut loops
reached new steady-state conditions after chyme had been removed.
Thereafter, an infusion of L-NMMA was begun into the jejunal artery catheter. The infusion rate was adjusted to achieve a
drug concentration of 104 M within arterial blood based
on the rate of blood flow at the time the drug infusion was begun.
Repeat measurements were obtained when steady-state conditions were
reestablished during the drug infusion.
Data Analysis
Quantification for both eNOS mRNA and protein were carried out on all harvested mesenteric artery samples. This approach allowed two statistical treatments of these data. First, the mean values of the mRNA copy number and densitometry signal from each age group were determined and compared among groups by ANOVA and post hoc Tukey's B tests. Second, a correlation between mRNA copy number and eNOS protein densitometry signal was carried out within each subject in each age group. The hemodynamic studies were carried out on separate groups of animals from those used for the mRNA and protein studies; therefore, direct statistical correlation between eNOS expression parameters and hemodynamic variables could not be carried out within individual subjects. Hemodynamic data were analyzed by ANOVA and post hoc Tukey's B tests. Statistical significance for all analyses was set at P < 0.01. All data are presented as means ± SD. ![]() |
RESULTS |
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Condition of the Postnatal Day 1 Subjects
The condition of the nonfed and fed subjects was similar at the time of study; indeed, it was not possible to differentiate between the two by outward physical appearance or behavior. Glucose, sodium, and blood gas data were similar in both subgroups (Table 1). Both subgroups had full bladders [volume 32 ± 7 ml, 270 ± 23 mosM (nonfed) vs. 41 ± 5 ml, 254 ± 12 mosM (fed)] and appeared well hydrated as evidenced by moist mucus membranes. Stomachs of the nonfed group contained a thin yellow fluid (weight of contents 7 ± 2 g; weight of cleansed stomach 23 ± 4 g), whereas the intestines contained a modest amount of clear, more viscous liquid (weight of contents 11 ± 3 g; weight of cleansed gut 112 ± 7 g). The stomachs of the fed group were distended with a large amount of white curd (weight of contents 73 ± 6 g; weight of cleansed stomach 32 ± 6 g), whereas their intestines contained abundant yellow chyme (weight of chyme 119 ± 6 g; weight of cleansed gut 154 ± 12 g).
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eNOS Expression Data
eNOS mRNA copy numbers in homogenates of mesenteric artery were remarkably stable throughout the entire age range studied (Table 2). The average mRNA copy number was not statistically different among any of the age groups. The mean of all subjects from all age groups, including both the day 1 nonfed and fed subgroups was 4.02 × 106 copies eNOS mRNA/ng total RNA. eNOS protein expression significantly changed within the time frame studied. Expression was relatively low in the fetal subjects, as well as in the 1-day-old nonfed and 30-day-old groups compared with the other age groups (Figs. 2, 3, and 4). A nearly twofold difference in eNOS protein expression was observed between the 1-day-old nonfed and fed subgroups, with the fed subgroup demonstrating more eNOS (Figs. 2 and 4). It is important to note that tissue harvest from these subgroups was carried out at the same postnatal age, i.e., ~20 h after birth, so that the sole difference among these subgroups was the presence or absence of feeding. Expression of eNOS protein increased further on postnatal day 10 but then receded to the fetal level by postnatal day 30 (Figs. 3 and 4). Statistical correlation between the mRNA copy number and densitometry data did not attain significance; indeed, the r2 for all subjects was 0.13 with a P value of 0.562. Greater significance was not achieved by generated correlation coefficients within each age group.
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Hemodynamic Data
The baseline hemodynamic conditions were significantly different among age groups (Tables 3 and 4). The blood flow rate into the gut loop was low in 1-day-old nonfed subjects compared with the 1-day-old fed group or the 3- or 10-day-old group. These differences were similar, regardless of the means used to express the flow rate, i.e., as a function of tissue weight (Table 3) or simply as milliliters per minute (Table 4), inasmuch as the tissue weight of these four groups were similar (Table 4). Changes in gut vascular resistance paralleled the changes in flow rate. Gut oxygen uptake was significantly lower in the 1-day-old nonfed subjects than in the 1-day-old fed group. The lower uptake in the unfed group reflected both a lower oxygen delivery (as evidenced by the lower flow rate) as well as a decreased (a-v)O2. Oxygen uptake continued to increase on postnatal days 3 and 10 but then significantly decreased on postnatal day 30. Significant differences in the response to L-NMMA were also noted among the age groups studied (Fig. 5). Subjects in the day 1 nonfed group had only a modest increase (12 ± 3%) in vascular resistance during L-NMMA infusion, and the final level of resistance was not significantly different from baseline. In contrast, subjects in the day 1 fed group demonstrated a brisk increase in vascular resistance (72 ± 4%) as did subjects in the 3- and 10-day-old groups (84 ± 5% and 66 ± 5%, respectively). The response of the 30-day-old group was statistically similar to the 1-day-old nonfed group; thus an increase of only 6 ± 2% was noted, which was not statistically different from baseline.
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DISCUSSION |
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The novel observation made in this study was that eNOS expression within the mesenteric artery changed within the time frame studied. This change occurred at the level of eNOS protein expression; thus eNOS mRNA remained stable, whereas eNOS protein was significantly greater in the day 1 fed subjects, as well as in the day 3 and 10 subjects, compared with the other age groups.
Expression of eNOS protein was significantly different between the postnatal day 1 fed and nonfed subgroups. A concern in study design was that the nonfed subjects would deteriorate during the 18-h period of fasting, i.e., between birth and study, because of lack of water and nutrient intake. This concern proved unfounded, however, as the fed and nonfed subgroups were physically indistinguishable at the time of tissue harvest; as well, both subgroups were euglycemic, well-hydrated, and had blood pressures within the range previously described for 1-day-old swine (3). The robust condition of the nonfed subgroup most likely reflects the accumulation of extensive water and carbohydrate stores during late fetal life that occurs in many mammalian species in preparation for birth (28). We thus interpret these data to indicate that the upregulation of eNOS protein evident in the fed subgroup occurred because of the initiation of postnatal feeding, rather than because subjects in the nonfed subgroup were ill. This conclusion might be strengthened by the addition of a third subgroup on postnatal day 1 that were fasted for 18 h after birth and then allowed to feed. However, it would be necessary to fast the relevant control for this additional group beyond 18 h of postnatal life, a process disallowed by the Institutional Animal Care and Use Committee governing this project. Although a linkage between the onset of postnatal feeding and upregulation of eNOS in the mesenteric artery can be established from the present data, this linkage cannot be interpreted as a direct cause-effect relationship between these events. Indeed, it is difficult to speculate as to a mechanism whereby absorbed nutrients would directly affect eNOS expression within intestinal endothelial cells, either in the intramural microvasculature or the more distal conduit vessels studied here. How then might the two events, the onset of postnatal feeding and the expression of eNOS protein in the mesenteric artery, be related?
One possibility is that the upregulation of eNOS within the mesenteric artery was stimulated by the increased rate of intestinal blood flow that occurred after the initiation of feeding, i.e., by the first postprandial hyperemia experienced by the intestine after birth. Postprandial hyperemia is the increase in gastrointestinal blood flow that follows feeding (5). Several vascular effector systems have been implicated in the generation of postprandial hyperemia, including gut peptides (29), putative metabolic feedback signal (14), PGI (4), and adenosine (32). The process is initiated within the intramural intestinal microcirculation, i.e., that portion of the intestinal circulation in close contact with absorbed nutrients; thus a reduction of downstream resistance initiates a net increase in the rate of intestinal blood flow (14). The composition of the meal is an important factor in determining the magnitude of postprandial vasodilation; thus fat serves as an exceptionally potent stimulus (34). This fact is particularly important here insofar as the fat content of sow's milk in the first postnatal week is exceptionally high (12) and has been shown to induce a more substantial postprandial response than carbohydrates or protein in newborn swine (7). In this latter context, it is important to note that nutrient-induced intestinal hemodynamic changes have been observed in very early newborn swine and that increases in both mucosal blood flow (6, 7, 26) and (a-v)O2, or oxygen extraction (8, 26), occur. Furthermore, it was clear during the hemodynamic protocol in this study that the gut loops in the postnatal day 1 fed subgroup were in a postprandial hemodynamic state when first created because the loops contained chyme; moreover, its removal caused a ~25% reduction in flow rate, a change consistent with the magnitude of the postprandial hyperemia in newborn swine (6, 26). We thus propose that the mesenteric artery experienced an increase in flow rate consequent to a downstream, nutrient-induced vasodilation that occurred on the initiation of postnatal feeding. We speculate that this increase in flow rate served as a stimulus for the upregulation of mesenteric artery eNOS, because expression of eNOS increases in response to the mechanostimulus of flow or shear stress (35, 38).
Whereas the aforementioned speculation may explain the upregulation of eNOS after birth, the basis for the subsequent reduction of eNOS expression within the mesenteric artery on postnatal day 30 remains an enigma. The diet in this oldest age group was substantially different from that experienced by the younger age groups, because it was entirely grain-based; moreover, the frequency of feeding in 30-day-old swine is substantially less (i.e., twice daily) than in younger piglets who feed 9 to 12 times a day, especially during the first postnatal week (2, 12). Yet is unclear how these differences might affect expression of eNOS within the mesenteric artery. Additional studies might focus on the effects of feeding on the degree of intestinal oxygenation or its redox state as putative factors that might affect eNOS expression.
Another interesting facet of the eNOS expression data is that the change in expression only occurred at the posttranscriptional level, i.e., that eNOS mRNA was stable between late fetal life and postnatal day 30. The present data do not provide a sufficient basis for speculation as to why only a change in protein expression was noted. Stability of eNOS transcription might be seen to compromise our speculation regarding the relationship between the postprandial flow rate and eNOS expression in mesenteric artery, insofar as observations linking shear stress and eNOS expression demonstrate an increase in both eNOS mRNA and protein (35, 38). However, the flow applied to these cultures was laminar, not pulsatile. Observations made in this study were generated within mesenteric artery exposed to a pulsatile flow, as evidenced by the substantial pulse pressure (23 ± 3 mmHg) recorded from the jejunal artery catheter. In this context, it is important to note that oscillatory shear stress has little impact on the rate of eNOS transcription within cultured endothelial cells (37). The mechanistic basis for the increased translation of eNOS without a concomitant change in transcription remains to be determined.
The present study expands previous observations from this laboratory regarding the postnatal intestinal circulation by adding additional age groups, especially the 1-day-old nonfed and fed subgroups. Our observation that intestinal blood flow and tissue oxygen uptake are relatively low and that vascular resistance is relatively high on the first postnatal day before feeding is consistent with studies by Crissinger and Burney (6-8), who first reported intestinal hemodynamics in 1-day-old piglets fasted from birth onward. These investigators did not compare nonfed and fed subjects at the same time on the first postnatal day, as was carried out here, although the substantial increase in baseline intestinal blood flow and oxygen uptake by postnatal day 3 and its decline by postnatal day 30 was first described by these workers (6-9). It is clear that baseline hemodynamic and oxygenation parameters dramatically change during early postnatal life in swine, a process also noted in other species (11, 16).
Previous reports (19-21, 25) from this laboratory have indicated that the relative role of NO in regulation of the postnatal intestinal circulation is age specific, being more dominant during very early newborn life. Present data confirm these past observations and provide a mechanistic basis for the greater constitutive (1, 19, 23) and stimulated (24, 30, 31) production of NO within the newborn intestinal circulation, i.e., that expression of eNOS within the mesenteric artery increases after birth and remains elevated for at least the first 10 days of postnatal life. It is critical to note, however, that the present study is insufficient to allow a correlation between the level of eNOS expression and intestinal vascular resistance. We are only able to conclude that the expression of eNOS changed within the mesenteric artery during early postnatal life, as did intestinal vascular resistance; a cause and effect linkage between these independent observations cannot be established from these data. It will be necessary to carry out studies in which the variables of flow rate, wall shear stress, vascular resistance, eNOS expression, and NO production are simultaneously measured in the same vessel before any inference or conclusions can be made regarding the potential interactions between the level of eNOS expression and vascular resistance.
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ACKNOWLEDGEMENTS |
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We thank Jim Miller and Hans Wooster, owners of the swine breeding farm in Baltimore, OH, where this study was initiated. Their steadfast cooperation in providing precise times of birth and removing piglets from the sow allowed this study to occur.
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FOOTNOTES |
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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-58580 (to K. M. Reber) and National Institute of Child Health and Human Development Grant HD-25256 (to P. T. Nowicki)
Address for reprint requests and other correspondence: P. T. Nowicki, Children's Hospital, 700 Children's Drive, Columbus, OH 43205 (E-mail: nowickip{at}pediatrics.ohio-state.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpgi.00067.2002
Received 25 February 2002; accepted in final form 13 May 2002.
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