Department of Pediatrics, The Ohio State University and The Vascular Biology Laboratory, Children's Research Institute, Children's Hospital, Columbus, Ohio 43205
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ABSTRACT |
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We studied mesenteric arterial
arcades from 3- and 35-day-old swine to determine the relationship
between perfusate flow rate and release of nitric oxide (NO) into
mesenteric effluent. Mesenteric arterial arcades were perfused under
controlled-flow conditions with a peristaltic pump using warm
oxygenated Krebs buffer. Basal rates of NO production were 43.6 ± 4.2 vs. 12.1 ± 2.5 nmol/min in 3- vs. 35-day-old mesentery during
perfusion at in vivo flow rates (9 vs. 20 ml/min, respectively). Rate
of NO production was directly related to flow rate over a wide range of
flows (5-40 ml/min) in 3- but not 35-day-old mesentery. Both age
groups demonstrated a brisk, albeit brief, increase in NO production in
response to infusion of NO-dependent vasodilator substance P
(108 M/min). Tyrosine kinase inhibitor herbimycin A and
L-arginine analog L-NMMA significantly
attenuated flow-induced increase in NO production, and phosphatase
inhibitor phenylarsine oxide increased magnitude of flow-induced
increase in NO production in 3-day-olds. Removal of extracellular
Ca2+ and depletion of intracellular Ca2+ stores
(Ca2+-free Krebs with EGTA plus thapsigargin) had no effect
on NO production in either group. Thus, basal rate of NO production is
greater in mesenteric arterial arcades from 3- than from 35-day old
swine, a direct relationship between flow rate and NO production rate is present in mesentery from 3- but not 35-day-olds, and
phosphorylation events are necessary for this interaction to occur.
phosphorylation; calcium; flow-induced dilation; newborn intestinal circulation
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INTRODUCTION |
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HEMODYNAMIC CONDITIONS
WITHIN early postnatal swine intestine are substantially age
dependent (17). Most importantly, resting vascular
resistance is much lower in newborn intestine, i.e., 0.60 vs. 1.65 mmHg · ml1 · min
1 · 100 g in 3- vs. 35-day-old subjects, respectively. This difference is the
consequence of factors intrinsic to the intestinal circulation itself,
inasmuch as they are equally present in denervated, blood-perfused in
vitro intestinal loops and innervated, autoperfused in situ intestine.
An intrinsic vascular regulatory system that appears to play an
age-specific role in regulation of intestinal vascular resistance is
nitric oxide (NO) produced by the endothelial isoform of NO synthase
(eNOS). Two lines of evidence support this contention. First, blockade
of endogenous NO production results in a significantly greater increase
in intestinal vascular resistance in 3- than in 35-day-old subjects
under both in vivo and in vitro conditions (15). Second,
relaxation in response to some NO-dependent vasodilators is of greater
magnitude in phenylephrine (PE)-precontracted mesenteric artery
rings from 3- than from 35-day-old swine. One interpretation of
these observations is that the newborn gut vasculature produces more NO
than does its older counterpart; however, direct measurement of NO
production by this system has not yet been carried out, and existing
data provide only indirect estimates.
The most potent and likely the most physiologically relevant stimulant of endothelial constitutive NOS (ecNOS) activity is the movement of blood against the static endothelial cell surface, an action that creates a measurable force called shear stress (9). Increased NO production in response to a flow stimulus has been demonstrated using cultured endothelial cells (3, 10) as well as in femoral artery segments perfused in vitro with Krebs buffer (19). Two experimental formats have been used in these studies. The first is comparison of the NO concentration within conditioned medium from endothelial cells grown in static culture with the concentration of NO within the effluent of medium perfused across these cells by means of a parallel plate flow apparatus (10). In the second format, NO production by cultured (10) or in situ (19) endothelial cells has been compared during perfusion at basal flow rate vs. an increased flow rate. To the best of our knowledge, no one has demonstrated the effect of flow reduction on endothelial NO production. The change in NO production following a flow increase occurs very rapidly (3, 10, 19), as does the physiological response to this action, called flow-induced dilation (11). The mechanism responsible for flow-induced eNOS stimulation differs from that noted following agonist-induced stimulation of the enzyme, e.g., by agents such as substance P or bradykinin. Thus flow-induced eNOS activation appears to be largely independent of Ca2+ flux but instead entails phosphorylation of eNOS and/or other proteins (1, 6).
The goal of these experiments was threefold: 1) to compare the basal, or constitutive, rate of NO production by the mesenteric arterial vasculature in 3- and 35-day-old subjects, 2) to determine the effects of a change in flow rate on NO production and compare this effect to that generated by agonist-induced eNOS stimulation by substance P, and 3) to determine if these responses are dependent on Ca2+ and/or tyrosine phosphorylation. To this end, sections of swine mesentery were buffer perfused in vitro at different flow rates and the concentration of NO was determined in the effluent. Studies were carried out following blockade of endogenous NO production, tyrosine kinase or phosphatase activity, and in a nominally Ca2+-free environment following depletion of intracellular Ca2+ stores and removal of Ca2+ from the buffer perfusate. Our observations suggest that the relationship between flow rate and NO production within postnatal mesenteric arteries is significantly age dependent, being far greater in newborn subjects.
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METHODS |
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Care and Handling of Experimental Animals
Two age groups were studied: 3-day-old (newborn, age range 1-4 days) and 35-day-old (weanling, age range 30-40 days). Subjects were fed an age-appropriate diet until 12 h before the study, when food, but not water, was withheld. Anesthesia was initiated with xylazine (5 mg/kg) and telezol (7.5 mg/kg), both given intramuscularly, and was maintained with pentobarbital sodium (5 mg/kg) given intravenously. Euthanasia was carried out following tissue removal by the administration of Succumb (1 ml/kg iv), given while the subjects were still anesthetized. Animal care was provided in accordance with the Guide for the Care and Use of Laboratory Animals (DHHS publication 85-234), and experimental protocols were approved by the Institutional Animal Care and Use Committee of the Children's Research Institute.Tissue Preparation
Subjects were anesthetized, and ventilation was initiated to maintain normal blood gas tensions. The small intestine was exposed, and the distal jejunal ileum was identified. The mesenteric artery was cannulated at the midpoint between its aortic origin and terminal ileal conclusion, so that the final one-third of the mesentery and intestine was distal to the catheter orifice within the vessel. An infusion of warm (38°C) oxygenated (16% O2-5% CO2-balance N2) Krebs buffer was begun at a flow rate of 5 ml/min. The composition of the Krebs buffer was, in mM: 118.1 NaCl, 4.5 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25.0 NaHCO3, 11.1 glucose, and 0.026 EDTA. The intestine was cut away from the mesentery at its insertion site along the entire length of the buffer-perfused segment, which was demarcated by its pale color, in contrast to the remainder of the blood-perfused intestine. Thereafter, the mesentery that had perfused the distal small intestine was cut away from the intestine and transferred to the perfusion apparatus.Perfusion Apparatus
The tissue was placed into a water-jacketed dish that had a large drainage port at the bottom that was covered with fine wire mesh. The dish was covered with plastic wrap to minimize heat and evaporative loss, and a temperature probe within the dish monitored tissue temperature. The mesenteric artery cannula was directed to a perfusion system that consisted of a buffer reservoir, a pump (Gilson Miniplus 4), and a heat exchanger. An electromagnetic flowmeter (Gould, 2.0-mm ID) and a standard pressure transducer were placed within the perfusion circuit. The effluent drained from the mesentery by gravity through the hole in the bottom of the glass dish and was diverted into a separate collection beaker. Buffer perfusion was carried out in a single-pass, or nonrecirculating, mode.Stabilization of the Preparation and Confirmation of Tissue Viability
The mesentery was perfused at 3 ml/min for 5 min to allow the tissue to warm. Flow was then increased to an age-appropriate baseline level that duplicated in vivo conditions. These flow rates were determined in preliminary studies by placing a cuff-type electromagnetic flow transducer on the mesenteric artery in situ at the site where cannulation would take place were the mesentery to be removed for in vitro perfusion. These rates were 9 and 20 ml/min for 3- and 35-day-old subjects, respectively. Two tests of preparation viability were carried out: contraction in response to PE (10Measurement of NO Production
NO is rapidly oxidized to NO2Considerations Necessary to Allow Comparison of NO Production Rates Within and Between Age Groups
The diameter and length of the mesenteric arterial vessels and hence the total endothelial cell mass was greater in older subjects, which in turn meant that direct comparison of endothelial NO production rates between age groups would be inaccurate unless some means to correct for this difference was applied. Simple correction based on the weight of the mesentery was not acceptable because other nonvascular tissue, particularly lymph nodes, made up a much larger percentage of the mesentery weight than did the blood vessels. Thus a correct factor based on vessel dimension was established. Swine have a single mesenteric artery trunk arising from the aorta, and the final one-third of this artery, which supplies the distal jejunum and ileum, is remarkably similar from subject to subject and was the portion selected for study (Fig. 1). This distal mesentery consists of four distinctive vessel types: 1) the main mesenteric artery, from which arise 2) a series of very short branches which feed into 3) an arterial plexus, from which arise 4) terminal mesenteric arterioles (TMA). TMA extend unbranched from the arterial plexus to the intestinal wall and constitute the overwhelming bulk (>90%) of the distal mesenteric arterial arcade; thus the correction factor was determined solely on the basis of the dimensions of the TMA without consideration of differences in size of the other three vessel types. A morphometric analysis was carried out in vivo in a separate group of subjects to measure TMA length and diameter (Table 1), and on the basis of these data the ratio of TMA surface area was calculated between age groups as 1:2.7.
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Experimental Protocols
Protocol 1. The goal of this protocol was twofold: first to compare the rates of NO production during perfusion at age-appropriate flow rates between groups and second to determine if manipulation of flow rate altered the rate of NO production within each group. Mesentery preparations were first perfused at the age-appropriate rate (9 vs. 20 ml/min); thereafter, flow rate was reduced to 5 ml/min and increased in stepwise increments to 10, 20, and 40 ml/min in both age groups. Each new flow was maintained until the measured variables attained new steady-state values; this time period was determined to be 3 min in pilot experiments.
Protocol 2.
The goal of this protocol was to determine the effects of endogenous NO
synthesis blockade
[N-monomethyl-L-arginine
(L-NMMA), 0.1 mM], tyrosine kinase inhibition (herbimycin
A 5 µM), phosphatase inhibition (phenylarsine oxide 10 µM), or a
nominal Ca2+-free environment on the changes in vascular
resistance and NO production rate caused by a single change in flow
rate. To this end, measurements were first carried out while the
mesentery was perfused at an age-appropriate flow rate and again after
a twofold increase or 50% reduction in this rate (e.g., from 9 to 18 ml/min or from 9 to 4.5 ml/min in the newborn group). The nominal
Ca2+-free environment was achieved by using
Ca2+-free Krebs buffer with 1 mM EGTA and 1 µmol
thapsigargin to deplete intracellular Ca2+ storage sites.
Drugs were added to the perfusate buffer 10 min before the onset of the
protocol, during which time new, postdrug steady-state pressure was
noted in all instances.
Protocol 3.
The goal of this protocol was to measure NO production and mesenteric
vascular resistance in response to the NO-dependent vasodilator peptide
substance P to allow comparison between agonist-induced and
flow-induced NO production. Substance P was continuously infused into
the perfusion circuit at a drug infusion rate set to attain a perfusate
concentration of 108 M. The infusion rate was
consistently 0.5 ml/min.
Statistical Analysis
The NO production rate was determined by multiplying the NO concentration (µM) within each 30-s effluent collection by the flow rate present during collection. The NO production rate for older subjects was adjusted by applying the correction factor of 2.7. Each data set was analyzed by means of a two-way ANOVA that used age group (3- vs. 35-day) and time (or change in flow rate) as main effects. Post-hoc Tukey B tests were carried out if the F-statistic for the ANOVA was significant (P < 0.01) to determine the specific sites of significance. ![]() |
RESULTS |
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The NO production rate observed under baseline conditions was age
dependent, being 41 ± 5 nmol/min at the age-appropriate baseline
flow rate of 9 ml/min in mesentery from 3-day-old subjects and 11 ± 4 nmol/min at the age-appropriate baseline flow rate of 20 ml/min in
the older group (means ± SD; P < 0.01;
n = 42). Vascular resistance was 2.23 ± 0.12 vs.
2.01 ± 0.16 mmHg · ml1 · min
1 at these
age-appropriate flow rates. Comparison of these resistance data to
those we have previously reported must be done with caution, because
the mesentery preparation has a distal pressure of zero (i.e., the cut
end of the TMA are open to the environment) and the data are not
expressed as a function of intestinal weight. The NO production rate
increased and vascular resistance decreased in response to step
increases in flow rate in mesentery removed from 3-day-old subjects
(Fig. 2). In contrast, manipulation of flow rate did not significantly affect the NO production rate or
vascular resistance in mesentery removed from 35-day-old subjects. Also, the NO production rate was consistently greater in the younger age group at all flow rates tested.
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NO production rate increased and vascular resistance decreased in
response to a single twofold increase in flow above the baseline rate
in 3- but not 35-day-old subjects (Figs.
3 and 4). The tyrosine kinase inhibitor herbimycin A and the
L-arginine analog L-NMMA significantly
attenuated the changes noted in 3-day-old mesentery; also,
L-NMMA significantly reduced the NO production rate noted
under baseline conditions. The phosphatase inhibitor phenylarsine oxide
increased the NO production rate following flow increase but had no
effect on vascular resistance. Removal of Ca2+ from the
perfusate and concomitant depletion of intracellular Ca2+
stores had no effect on the rate of NO production or vascular resistance. In contrast to the effects noted in the newborn group, none
of the experimental treatments altered the effect of a twofold flow
increase on NO production or vascular resistance in mesentery from
35-day-old subjects.
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NO production decreased following a 50% reduction in the
age-appropriate baseline flow rate in 3- but not 35-day-old mesentery, although flow rate reduction had no effect on vascular resistance (Figs. 5 and
6). L-NMMA decreased the
baseline rate of NO production and eliminated the flow-induced
reduction in this variable in younger subjects but had no effect on
vascular resistance. Phenylarsine oxide also eliminated the
flow-induced reduction in the NO production rate but again had no
effect on vascular resistance in 3-day-old mesentery. Neither a nominal
Ca2+-free environment nor herbimycin A significantly
altered the flow-induced changes in the NO production rate or vascular
resistance in mesentery from newborn subjects. A 50% reduction in the
age-appropriate flow rate had no effect on the rate of NO production or
vascular resistance in 35-day-old mesentery, and this response was not affected by any of the experimental treatments.
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The addition of substance P to the perfusate caused an increase in the
rate of NO production and vasodilation to a similar magnitude in both
age groups (Fig. 7). The response
occurred rapidly, but, despite a continued drug infusion, the effect
waned quickly such that the measured variables had reverted to their
respective baseline values within 2 min after the onset of peptide
infusion. The relative magnitude of change in the NO production rate
was similar in newborn mesentery in response to a flow stimulus or substance P, except that the former effect was sustained and the latter
was not (i.e., compare Figs. 2 and 7). In contrast to the younger
group, the magnitude of change in the NO production rate was
substantially greater in response to substance P than in response to
flow.
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DISCUSSION |
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The Basal Rate of NO Production by Swine Mesentery is Age Dependent
The basal NO production rate, as assessed by measurement of NO in the effluent of buffer-perfused mesenteric arterial arcades during constant-flow, peristaltic perfusion at flow rates that duplicated in vivo conditions was greater in younger subjects, and this difference was maintained or increased at all flow rates tested. Although the difference was substantial, between-group comparison must be made with caution. First, the correction factor of 2.7 applied to the NO production rate in older subjects was only an approximation of the difference in endothelial surface area between age groups. It is noteworthy, however, that a significant difference between groups was present before the correction factor was applied and that the surface area of the mesenteric arterial arcade was clearly greater in older subjects; indeed, the correction factor likely underestimates the difference. Also, a similar outcome was noted when the NO production data were simply normalized to wet or dry tissue weight; indeed, the difference between age groups increased under these circumstances, because the mass of the 35-day-old mesentery was substantially greater than that from 1-day-old subjects. Second, the viscosity of the buffer perfusate, an important component of shear stress, was substantially less than that of blood. It is important to note, however, that the hematocrit, serum protein concentration, and whole blood viscosity are similar in 3- and 35-day-old swine (4), so that the viscosity term would have been equal between groups under more relevant in vivo conditions. Third, measurement of NO released into the effluent as an index of the NO production rate by endothelial cells might be challenged. NO carries out its hemodynamic task by rapidly diffusing to vascular smooth muscle on the abluminal side of the endothelial cell (12); as such, NO released into the vascular space might be more representative of spillover rather than being a true index of physiologically relevant NO production by mesenteric arterial endothelial cells. Within the constraints placed by these caveats, we conclude that the amount of NO released from buffer-perfused mesenteric arterial tree is greater in newborn subjects.The conclusion that newborn mesenteric vessels maintain a greater constitutive rate of NO production under basal flow conditions is consistent with previous observations from this laboratory. Thus we have demonstrated that blockade of endogenous NO synthesis with L-NMMA in vivo causes a significantly greater degree of vasoconstriction in 3- than in 35-day-old subjects and that the response of PE-precontracted mesenteric artery rings to NO-dependent vasodilators is greater in younger subjects (15). We have previously proposed that the newborn intestinal circulation differs from its adult counterpart and that an important basis for this difference is that eNOS-derived NO plays a greater role in setting basal vascular tone in younger animals (15). The present data support this contention.
Effect of a Change in Flow Rate on NO Production is Age Dependent
A direct relationship between flow rate and NO production rate was present in mesenteric arterial arcades from 3- but not 35-day-old swine, but both age groups demonstrated a significant increase in NO production in response to the peptide agonist substance P. Substance P exerts its vascular effect by binding to endothelial NK1 receptors. Ligand binding to these serpentine G protein-linked receptors leads to activation of phospholipase C and subsequent production of inositol triphosphate and diacylglycerol; hence, this action increases cytosolic Ca2+, which ultimately activates eNOS. In contrast, eNOS activation by the mechanostimulus of flow or shear stress does not appear to be contingent on Ca2+ (1, 6) but instead appears to involve serine phosphorylation of eNOS (5, 7). The present data are consistent with these previous observations and suggest that the age-dependent effect of flow rate on NO production in postnatal swine mesentery is consequent to factors upstream from eNOS, possibly in the mechanotransducer or subsequent signal transduction pathway whose endpoint is phosphorylation of eNOS or related proteins.Of great interest is the observation that the NO production rate decreased in response to a reduction in the flow rate in newborn mesentery. To the best of our knowledge, this process has not been demonstrated in other circulations. A great deal of work has been carried out to delineate the mechanistic basis for stimulation of ecNOS activity in response to an increased shear stress (1, 5, 6, 14); to date, the reverse process has received little attention and the present data are insufficient to generate speculation as to the mechanism. The observation might have substantial significance in local regulation of vascular resistance because the physiological actions of NO transcend its cGMP-mediated direct effect on vascular smooth muscle tone. NO inhibits the functional activity of locally produced constrictor agents such as angiotensin (2, 21) and endothelin-1 (21); in some instances, this effect is mediated by the ability of NO to interfere with receptor-G protein coupling (13, 20). Thus a reduction of flow rate through the intestine, e.g., caused by systemic hypotension, would lower the constitutive rate of NO production, directly decreasing the NO vasodilator stimulus and indirectly increasing the potency of vasoconstrictor agents within the gut microcirculation. These events might rapidly amplify an otherwise trivial ischemic event. In this context, it is interesting to note that the newborn intestinal circulation is uniquely susceptible to rapidly progressive vasoconstriction in response to hypotension (18).
Flow-Induced Increase in NO Production is Dependent on Tyrosine Phosphorylation Events
As noted, flow-induced activation of ecNOS was partly (10) or completely (1, 7) independent of Ca2+ but instead is consequent to phosphorylation events within the endothelial cell. Thus work by Fleming et al. (6) and Corson et al. (3) demonstrated tyrosine phosphorylation of endothelial proteins in response to a shear stimulus. More recently, serine phosphorylation of eNOS in response to a flow stimulus by the protein kinase AKT has been reported by Fulton et al. (7) and Dimmeler et al. (5). Observations made in these experiments are consistent with these previous reports: the tyrosine kinase inhibitor herbimycin A decreased the flow-induced increase in NO production in newborn mesentery, whereas the phosphatase inhibitor phenylarsine oxide enhanced this response. Phosphorylation of eNOS was not measured in these experiments, and it is unclear which protein(s) was affected by kinase or phosphatase inhibition. We can only suggest that the signal transduction pathway by which the mechanostimulus of shear stress activates ecNOS includes protein phosphorylation. Furthermore, the present data strongly suggest that the age-specific difference lies upstream from eNOS itself, inasmuch as substance P activation of the enzyme was similar in both age groups.In conclusion, three novel observations were made in this study: 1) the basal, or constitutive, rate of NO production in buffer-perfused mesenteric arteries is age dependent, being greater in younger subjects; 2) a direct relationship between flow rate and NO production rate is present in mesentery from 3- but not 35-day-old subjects; and 3) the flow-induced increase in NO production in newborn mesentery is contingent on tyrosine phosphorylation events. It appears that the age-dependent effect of flow on NO production is consequent to differences upstream from eNOS, possibly involving the mechanotransducer or subsequent signal transduction pathway.
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ACKNOWLEDGEMENTS |
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We wish to thank Dr. Craig Nankervis for a critical reading of this manuscript. Karen Watkins provided outstanding secretarial support in the completion of this project.
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FOOTNOTES |
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This work was supported by a grant from the Child Health Research Center (K. M. Reber) and Grant HD-25256 (P. T. Nowicki) from the National Institutes of Child Health and Human Development
Address for reprint requests and other correspondence: K. M. Reber, Children's Hospital, 700 Children's Dr., Columbus, OH 43205 (E-mail: reberk{at}pediatrics.ohio-state.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 27 December 1999; accepted in final form 15 August 2000.
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