Determinants of terminal mesenteric artery resistance during the first postnatal month

Craig A. Nankervis, David J. Dunaway, and Philip T. Nowicki

Vascular Biology Laboratory, Children's Research Institute, Children's Hospital, and Department of Pediatrics, College of Medicine and Public Health, The Ohio State University, Columbus, Ohio 43205


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Experiments were conducted to delineate the vascular effector systems that contribute to setting mesenteric vascular tone in swine during the first postnatal month. Terminal mesenteric arteries (TMA), which function as resistance vessels, were studied in vitro with a microvascular perfusion system allowing independent pressure and flow manipulation. When pressure was varied 0-100 mmHg in the absence of flow, TMA from 1-day-old animals demonstrated myogenic vasoconstriction, whereas TMA from 40-day-old animals did not. In 1- but not 40-day-old TMA, the endothelin A (ETA) receptor antagonist BQ-610 shifted the pressure-diameter curve upward, whereas the ETB receptor antagonist BQ-788 and the L-arginine analog NG-monomethyl-L-arginine (L-NMMA) shifted the curve downward; in all instances, myogenic vasoconstriction was preserved. Flow eliminated myogenic vasoconstriction in 1-day-old TMA, i.e., diameter increased as a function of pressure. The effect of BQ-610 was lost under flow conditions; however, BQ-788 and N-acyl-L-Trp-3,5-bis-(trifluoromethyl) benzyl ester, an antagonist specific to the substance P neurokinin-1 (NK1) receptor, shifted the pressure-diameter curve downward in the presence of flow, whereas L-NMMA restored myogenic vasoconstriction. Adding flow had no effect on the pressure-diameter relationship in 40-day-old TMA. Other blocking agents, including prazosin, losartan, indomethacin, and charybdotoxin, had no effect on the pressure-diameter relationship in either age group under flow or no-flow conditions. Constitutive production of nitric oxide (NO) and endothelin-1 participates in setting resistance in 1-day-old TMA, and important stimulants to NO production include flow and activation of ETB and NK1 receptors. In contrast, 40-day-old TMA act as passive conduits in which the elastic properties of the vessel are the primary determinant of diameter.

myogenic response; endothelin; nitric oxide


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE HEMODYNAMIC CHARACTERISTICS of terminal mesenteric arteries (TMA), an important part of the resistance vasculature in swine intestine, substantially change during the first postnatal month (27). TMA from 1-day-old swine constrict in response to increased intravascular pressure; however, this myogenic response is counterbalanced under normal hemodynamic conditions by a nitric oxide (NO)-dependent, flow-induced dilation. In contrast, TMA from 40-day-old swine demonstrate neither myogenic constriction in response to pressure elevation nor dilation in response to a flow stimulus. It thus appears that vascular tone in TMA is set by constitutively active, intrinsic vascular effector systems during early newborn life, whereas the passive elastic characteristics of the vessel are the principal determinants of diameter at the end of the first postnatal month.

Other vascular effector systems that might impact on the postnatal change in TMA hemodynamics have not been fully delineated. For example, it is established that blockade of endogenous NO synthesis with the L-arginine analog NG-monomethyl-L-arginine (L-NMMA) significantly decreases the diameter of 1-day-old TMA. However, loss of constitutive dilator tone will result in vasoconstriction only if a constitutive constrictor tone is present, waiting to be unmasked as the system is unbalanced. Underlying myogenic tone could be a contributing factor here, but it cannot fully account for this action as L-NMMA reduces 1-day-old TMA diameter even when intravascular pressure is below that necessary to engage the myogenic mechanism (27). A feasible candidate for this role is the constrictor peptide endothelin-1 (ET-1), which is produced in relatively large quantities by the newborn intestinal vascular endothelium (16, 32). The factors affecting the activity of the endothelial isoform of NO synthase (eNOS) in 1-day-old TMA have also not been determined. Thus, although eNOS activity is constitutive, Ca2+-calmodulin binding increases the rate of NO production (14), as does serine phosphorylation (5, 7). Substance P (SP), which exerts its vascular effect by increasing endothelial intracellular Ca2+ concentration ([Ca2+]i) (23, 24), and the mechanostimulus of flow, which causes NO-dependent dilation by phosphorylation of eNOS (5, 7), have both been demonstrated to affect resting intestinal vascular resistance in 1- but not 40-day-old swine and might therefore affect the basal activity of eNOS in younger TMA (17, 20).

The goal of these experiments was to further delineate factors responsible for setting basal vascular tone in 1- and 40-day-old TMA. We were particularly interested to determine the constrictor systems that, with the myogenic mechanism, set basal tone in 1-day-old TMA; we also wished to delineate the contributions of flow and nonflow stimuli to the basal or constitutive activity of eNOS. To this end, a variety of receptor, ion channel, and enzyme antagonists were administered to buffer-perfused TMA, and the effects of these agents on the pressure-diameter relationship were determined under both no-flow and flow conditions. Our observations indicate that resistance in 1-day-old TMA is determined by a balance among active constitutive forces, including myogenic tone, NO, and ET-1, and that the stimuli for NO production include the mechanostimulus of flow, as well as activation of ETB and neurokinin-1 (NK1) receptors by ET-1 and SP, respectively. In contrast, vascular resistance in 40-day-old TMA is not established by active constitutive forces but instead is primarily determined by the passive elastic characteristics of these vessels.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animal Acquisition and Handling

Studies were conducted on 1- and 40-day-old swine obtained from a local breeder on the day of use. The 1-day-old group was allowed to suckle after birth for ~12 h and was then fasted for 8 h before use. The 40-day-old group was also fasted for ~8 h before use. Anesthesia was induced with Telazol (6 mg/kg im) and xylazine (7.5 mg/kg im) and maintained with pentobarbital sodium (5 mg · kg-1 · h-1 iv). Animals were killed with an overdose of pentobarbital (5 mg/kg iv) while still anesthetized. All animal care was provided in accordance with the Guide for the Care and Use of Laboratory Animals [DHHS Publication No. (NIH) 85-23, Revised 1985, Office of Science and Health Reports, Bethesda, MD 20892] under the auspices of an experimental protocol approved by the Children's Research Institute Animal Care and Use Committee. All work involving live animals was carried out in the Children's Research Institute vivarium, an American Association for Accreditation of Laboratory Animal Care-approved facility under the supervision of a full-time veterinarian.

Experimental Preparation

Vessel removal. TMA represent the most distal branch of the mesenteric arterial arcade in swine. These vessels run unbranched from a dense vascular plexus to the wall of the intestine. Their in situ diameter and length are 180 µm and 2.7 cm, respectively, in 1-day-old swine and 300 µm and 4.8 cm, respectively, in 40-day-old swine. A significant pressure drop occurs from the origin to the endpoint of the TMA, a feature that characterizes them as resistance vessels in the mesenteric arterial arcade (27). TMA were removed from the experimental animals and immediately mounted in the proper proximal-distal orientation between two glass micropipettes seated within a plastic chamber (CH/2/AS, Living Systems, Burlington, VT). The inflow pipette was fixed, whereas the outflow pipette was mounted on a micrometer to allow adjustment along the longitudinal axis of the vessel. The vessel was secured in place by 11-0 ophthalmic suture.

TMA perfusion. Arterial pressure and flow were adjusted by means of a pressure-servo system (PS/200/Q, Living Systems), using a configuration previously described (27). Briefly, two pressure-servo devices were placed on either end of the TMA so that inflow pressure (P1) and outflow pressure (P2) could be independently manipulated. This arrangement allowed for discrete, separate manipulation of pressure and flow. Krebs buffer of the following composition was used as perfusate (in mM): 118.1 NaCl, 4.8 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 11.1 glucose, and 0.026 EDTA. The buffer was aerated with 16% O2-5% CO2, balance N2. At 38°C, the buffer pH was 7.40, pO2 was 85 mmHg, and pCO2 was 40 mmHg.

Vessel chamber suffusion. The vessel chamber and thus the exterior surface of the artery were continuously suffused with warm (38°C), aerated Krebs buffer. The suffusate buffer was recirculated at a rate of 50 ml/min, and the total suffusate volume was 200 ml. All vasoactive agents used in these experiments were added to the suffusate buffer reservoir in a final volume of 1 ml.

Measurement of TMA diameter, pressure, and flow rate. The chamber was mounted on the stage of an inverted microscope set in line with a video camera. Vascular dimensions were measured with a precalibrated video dimension analyzer (V94, Living Systems) that displayed wall thickness and luminal diameter. Flow rate across the TMA was measured with a flowmeter set in line with the perfusion system (Omega, Putnam, CT). P1 and P2 were measured with standard transducers.

Experimental Protocols

Initial stabilization. All vessels were kept at a pressure of 20 mmHg for 30 min after mounting. During this time, the suffusate buffer temperature was slowly increased from 23°C to 38°C. The pressure was then rapidly increased to 100 mmHg in 20-mmHg increments over 5 min to ensure the absence of vessel kinks and leaks. Thereafter, the pressure gradient across the artery was brought to zero by setting P1 and P2 at 45 mmHg in 1-day-old animals or 53 mmHg in 40-day old animals; these pressures duplicate the mean pressure within TMA in vivo (27). After a 30-min equilibration period, during which time all vessels developed spontaneous tone, the suffusion buffer was replaced with a 40 mM KCl-Krebs buffer. Vessels that failed to contract to 50% of their baseline diameter were discarded. The suffusion buffer was then replaced with standard Krebs buffer, and the experimental protocol was initiated.

Protocol 1. In this protocol, the relationship between pressure and vessel diameter was determined in the absence of flow, and the effects of several vasoactive blocking agents on this relationship were noted. To this end, P1 and P2 were simultaneously adjusted so that the pressure gradient across the TMA remained at zero. Pressure was first reduced to 0 mmHg and then increased in a stepwise fashion until a final pressure of 100 mmHg was attained; each pressure was maintained until TMA diameter reached a new steady-state level, defined as change in diameter <5% over 3 min. P1 and P2 were reset to age-appropriate baseline levels, and a single drug was added to the suffusion buffer after steady-state conditions were restored. Thereafter, the pressure ramp was repeated. The vasoactive blocking agents used in this protocol included 1 µmol prazosin (alpha 1-antagonist), 1 µmol losartan (AT1 antagonist), 5 µmol BQ-610 (ETA antagonist), 100 µmol L-NMMA (NOS inhibitor), 50 µmol indomethacin (cyclooxygenase inhibitor), 5 µmol BQ-788 (ETB antagonist), 0.5 nmol N-acyl-L-Trp-3,5-bis-(trifluoromethyl) benzyl ester (NATB, NK1 antagonist), and 10 µmol charybdotoxin (Ca2+-activated K+ channel blocker). The IC50 of these blocking agents was determined in this laboratory during the course of other studies (Refs. 15-17a and 20 and unpublished results). In all instances, the dose used in the present experiments was 10-fold greater than the IC50 to ensure effective blockade. The maximal passive diameter was determined in some vessels by removing Ca2+ from the perfusion and suffusion buffers and adding EGTA and 10 µmol papaverine before initiation of the pressure ramp. Some vessels received a combination of blocking agents, i.e., BQ-610 + BQ-788 or BQ-610 + L-NMMA.

Protocol 2. In this protocol, the relationship between pressure and vessel diameter was determined in the presence of flow, and the effects of several vasoactive blocking agents on this relationship were noted. The average flow rate through TMA under in vivo conditions is age specific, i.e., 50 and 100 µl/min for 1- and 40-day-old swine, respectively (27). Accordingly, flow rates of 50 and 100 µl/min were established across 1-day-old TMA, whereas rates of 100 and 200 µl/min were used in 40-day-old TMA. The use of two flow rates facilitated independent assessment of flow rate as a hemodynamic variable (as opposed to the simple presence of flow per se) and also allowed comparison between groups when perfused at a common flow rate, i.e., 100 µl/min. Each flow rate was established at mean arterial pressures of 20, 40, 60, and 80 mmHg by first setting P1 and P2 at equal pressures and then simultaneously increasing and decreasing P1 and P2, respectively, by identical amounts until the desired flow rate was achieved. We have previously demonstrated (27) that this approach maintains the mean pressure within the vessel at the starting level while establishing a pressure gradient and thus flow across the vessel. In some vessels, flow rates of 50 (for 1-day-old animals) and 100 (for 40-day-old animals) µl/min were established at the four different starting pressures before and after the addition of 5 µmol BQ-610, 5 µmol BQ-788, or 100 µmol L-NMMA. In preliminary studies, the effects of these agents were not different when TMA were perfused at a higher flow rate (i.e., 100 and 200 µl/min in 1- and 40-day-old animals, respectively) and so these additional studies were not pursued. Also, preliminary studies failed to demonstrate a significant effect of prazosin, losartan, indomethacin, or charybdotoxin on the pressure-diameter relationship under flow conditions.

Statistical Analysis

Data sets were initially analyzed using a three-way ANOVA for repeated-measures format. The main effects for the ANOVA were age group (1 vs. 40 day), condition (control vs. drug pretreatment), and time (i.e., different pressures). The F statistic for the three-way ANOVA was consistently significant (P < 0.01) for all data sets, as was the specific F statistic for the main effect of age group. This result indicated that the age groups acted in significantly different ways to the applied perturbations. To further delineate the sites of significant difference, subsequent two-way ANOVA was carried out on data sets within each age group using condition (control vs. drug pretreatment) and time (i.e., pressure) as main effects. If the F statistic for the two-way ANOVA was significant, then post hoc Tukey B tests were carried out to determine specific sites of significant difference.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The response to pressure and flow stimuli was substantially different in TMA from 1- and 40-day-old swine (Figs. 1 and 2). In the absence of intraluminal flow, vessel diameter decreased as pressure was raised between 40 and 80 mmHg in 1-day-old TMA, i.e., the vessels demonstrated myogenic vasoconstriction in response to stepwise incremental increases in pressure over the pressure range that brackets the normal mean arterial pressure at this postnatal age. In the presence of flow, however, an increase in vessel diameter occurred between 40 and 80 mmHg, replacing the myogenic vasoconstriction observed under no-flow conditions. This effect of flow was rate dependent; thus vessel diameter was greater at a flow rate of 100 vs. 50 µl/min when pressure was fixed at 40, 60, or 80 mmHg. In the absence of flow, the difference between the maximal diameter attained in Ca2+-free 10 µmol papaverine buffer and that noted in control vessels was 46 ± 4 µm when pressure was set at 60 mmHg, the normal mean arterial pressure at this postnatal age; in contrast, this difference was only 14 ± 5 µm in the presence of an intraluminal flow rate of 50 µl/min. TMA from 40-day-old swine did not demonstrate myogenic vasoconstriction but instead showed progressive dilation in response to stepwise incremental increases in pressure. The addition of flow had no effect on the pressure-diameter relationship. Also, the difference between the active and passive diameters at a pressure of 80 mmHg, the average mean arterial pressure at this postnatal age, was only 11 ± 3 µm, significantly less than that observed in 1-day-old TMA. The difference in the magnitude of response to pressure change noted in each age group could have resulted, in part, from the different resting diameters of these vessels. However, analysis after expression of the diameter as a function of the initial diameter at a pressure of 0 mmHg provided significance identical to that noted when nontransformed data were used.


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Fig. 1.   Relationship between pressure and vessel diameter in 1-day-old terminal mesenteric arteries (TMA) in the presence (constant flow rate of 50 or 100 µl/min) or absence of flow (no flow). Ca2+-free buffer contained 0.1 mmol EGTA and 10 µmol papaverine. Values are means ± SD; n = 5 for all observations. a P < 0.01 vs. F-0; b P < 0.01 vs. F-50.



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Fig. 2.   Relationship between pressure and vessel diameter in 40-day-old TMA in the presence (constant flow rate of 100 or 200 µl/min) or absence of flow (no flow). Ca2+-free buffer was as explained in Fig. 1 legend. Values are means ± SD; n = 5 for all observations. There were no significant differences among the 4 curves.

The effects of vasoactive blocking agents on the pressure-diameter relationship observed in the absence of flow were significantly different in 1- and 40-day-old TMA. In younger swine, the ETA receptor antagonist BQ-610 caused an upward shift of the pressure-diameter relationship, although the magnitude of difference between treated and control arteries was significantly greater only at pressures of 0 and 20 mmHg, which are below the pressure necessary to engage the myogenic response in these vessels (Fig. 3). BQ-610 did not affect the myogenic response between pressures of 40 and 80 mmHg, because the diameter of treated vessels consistently decreased in all experiments within this pressure range. The opposite effect was observed in TMA treated with either the ETB receptor antagonist BQ-788 or the L-arginine analog L-NMMA. Thus diameter was significantly lower in treated TMA than in control TMA across the entire range of pressures, and vasoconstriction was consistently observed when pressure was raised to 40, 60, and 80 mmHg. Simultaneous administration of BQ-610 and BQ-788 or BQ-610 and L-NMMA had no net effect on vessel diameter at pressures >20 mmHg, because the constriction caused by one agent was equally balanced by the dilation caused by the other; however, at pressures of 0 and 20 mmHg, a significant increase in vessel diameter was noted in the presence of both antagonists (Fig. 4). Prazosin, losartan, indomethacin, NATB, and charybdotoxin had no effect on the pressure-diameter relationship in 1-day-old TMA observed in the absence of flow (Table 1). None of the agents tested altered the relationship between pressure and diameter obtained in the absence of flow in TMA from 40-day-old swine (Fig. 5 and Table 1).


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Fig. 3.   Relationship between pressure and vessel diameter in 1-day-old TMA in the absence of flow after pretreatment with BQ-610 (5 µmol), BQ-788 (5 µmol), or NG-monomethyl-L-arginine (L-NMMA; 100 nmol). Values are means ± SD; n = 5 for all observations. a P < 0.01 vs. control.



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Fig. 4.   Relationship between pressure and vessel diameter in 1-day-old TMA in the absence of flow after pretreatment with BQ-610 (5 µmol) + BQ-788 (5 µmol) or BQ-610 (5 µmol) + L-NMMA (100 nmol). Values are means ± SD; n = 5 for all observations. There were no significant differences among the 3 curves.


                              
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Table 1.   Effects of blocking agents on relationship between pressure and vessel diameter in absence of flow in 1- and 40-day-old swine TMA



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Fig. 5.   Relationship between pressure and vessel diameter in 40-day-old TMA in the absence of flow after pretreatment with BQ-610 (5 µmol), BQ-788 (5 µmol), or L-NMMA (100 nmol). Values are means ± SD; n = 5 for all observations. There were no statistical differences among the 4 curves.

A different picture emerged in 1-day-old TMA when the blocking agents were administered in the presence of flow (Fig. 6). The dilatory effect of BQ-610 was only evident when pressure was set at 20 mmHg; at higher pressures, vessel diameter was similar in treated and control TMA. The constrictor effect of ETB receptor blockade remained intact as BQ-788 caused a downward shift in the pressure-diameter curve; however, the slope of the curve remained unchanged, i.e., myogenic vasoconstriction was still replaced by progressive dilation at each successively higher pressure. A similar trend was observed after blockade of NK1 receptors with NATB, i.e., a parallel, downward shift in the pressure-diameter curve was noted when the action of SP was blocked. In contrast to BQ-788 and NATB, however, NO synthesis blockade with L-NMMA restored the myogenic response as vessel diameter remained steady between 40 and 80 mmHg, despite the presence of a flow stimulus. The simultaneous addition of BQ-610 and BQ-788 or BQ-610 and L-NMMA caused an effect similar to that noted in response to singular blockade with BQ-788 or L-NMMA, respectively. Prazosin, losartan, indomethacin, and charybdotoxin had no effect on the pressure-diameter relationship obtained in the presence of flow in 1-day-old TMA (Table 2). None of the blocking agents tested altered the pressure-diameter relationship obtained in the presence of flow in 40-day-old TMA (Fig. 7 and Table 2).


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Fig. 6.   Relationship between pressure and vessel diameter in 1-day-old TMA in the presence of flow after pretreatment with BQ-610, BQ-788, N-acyl-L-Trp-3,5-bis-(trifluoromethyl) benzyl ester (NATB), and L-NMMA. All arteries were perfused at a constant flow rate of 50 µl/min. Values are means ± SD; n = 5 for all observations. a P < 0.01 vs. control; b P < 0.01 vs. value at 20 mmHg.


                              
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Table 2.   Effects of blocking agents on relationship between pressure and vessel diameter in presence of flow in 1- and 40-day-old swine TMA



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Fig. 7.   Relationship between pressure and vessel diameter in 40-day-old TMA in the presence of flow after pretreatment with BQ-610, BQ-788, and L-NMMA. All arteries were perfused at a constant flow rate of 100 µl/min. Values are means ± SD; n = 5. There were no statistical differences among the 4 curves.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

These observations were made in TMA, which we (27) have previously identified as an important part of the resistance vasculature in swine. However, it is important to understand a priori that the responses noted in these vessels may not be representative of the response of the entire intestinal circulation. A clear example of this point is evident in the response to a change in intravascular pressure noted in these vessels. Thus under no-flow conditions, we observed that an increase in transmural pressure caused a significant myogenic vasoconstriction in TMA from 1- but not 40-day-old swine. This finding is inconsistent with the observation of Crissinger and colleagues (4), who demonstrated that the magnitude of myogenic vasoconstriction in vascularly isolated gut loops was greater in older than in newborn piglets. However, the means by which the pressure stimulus was delivered (i.e., retrograde venous pressure elevation vs. increased P1) were different in the two studies (4). We chose the reductionist TMA model as a means to carefully control the experimental conditions and thus identify vascular control systems responsible for the observed changes. Interpretation and extrapolation of these observations must therefore be made within the context of the experimental model.

ET-1 Is a Determinant of Vascular Tone in 1- but Not 40-Day-Old TMA

The first novel observation made in these experiments is that ET-1 provides a constitutive constrictor tone in 1- but not 40-day-old TMA and that this effect is fully independent of the myogenic mechanism. Thus the integrity of myogenic vasoconstriction between pressures of 40 and 80 mmHg is not affected by singular or combined application of BQ-610 and BQ-788 as the pressure-diameter curves were either parallel or equal to the control curves over this pressure range. However, at pressures less than those necessary to engage the myogenic mechanism, i.e., <40 mmHg, the net constrictor effect of ET-1 was still apparent andstatistically more relevant than at higher pressures, where both the myogenic mechanism and ET-1-induced constriction contribute to vascular tone. Interestingly, the constrictor tone generated by ET-1 is almost precisely offset by a dilator tone generated by its binding to ETB receptors when combined blockade is administered at pressures of 40-80 mmHg, a range that brackets the pressures normally experienced by these vessels in situ (2, 8). The constrictor effect of ET-1 is mediated by binding to ETA receptors located on vascular smooth muscle (32). Ligand binding to these G protein-coupled receptors activates phospholipase C, causing generation of inositol trisphosphate and diacylglycerol, lipid mediators that increase [Ca2+]i and thus activate myosin light chain kinase (10, 29). The dilator effect of ET-1 is mediated by binding to ETB receptors located on endothelial cells (3, 30). The signal transduction pathway engaged by ligand binding to the ETB receptor is similar to that for the ETA receptor, except that elevation of [Ca2+]i activates eNOS to increase NO production (3, 30), which in turn exerts a dilatory tone. The presence of both receptor subtypes is clearly indicated in 1- but not 40-day-old TMA. This observation is consistent with the effects of exogenous ET-1 administration or ET receptor blockade in blood-perfused intestinal loops, which demonstrate the presence of ETA receptor function on postnatal days 1 and 30, but ETB receptor function only on day 1 (16).

The 1-day-old swine intestine generates a relatively large amount of ET-1, as evidenced by the presence of a greater concentration of the peptide in the mesenteric venous effluent than on the arterial side of the circulation (16). The reason for this robust ET-1 production is not known, although it seems feasible to speculate that its presence contributes to the angiogenesis and vascular remodeling that must be present within the intestine during early postnatal life (6, 12). The wet weight of the total small intestine averages 80 g in 1-day-old swine before the onset of suckling, whereas at the end of the first postnatal month, the weight is more than sevenfold greater, averaging 600 g. This rate of growth is disproportionate with respect to total body mass, as body weight only triples during the first postnatal month, from ~1.5 to ~4.5 kg in this species. This intense parenchymal growth requires continual expansion of the vascular infrastructure of the intestine, and ET-1 is an established proangiogenic factor. It thus seems logical to anticipate the existence of ETB receptors to provide a dilatory force that counterbalances the constrictor force generated by ETA receptors in 1-day-old intestine, thus allowing a substantial presence of ET-1 without creating unbalanced vasoconstriction.

Multiple Factors Contribute to Activation of eNOS in 1-Day-Old TMA

The second novel observation made in these experiments is that eNOS activity is modified by several factors in 1-day-old TMA. eNOS is a constitutive enzyme, i.e., a basal level of endothelial cell NO production is consistently present (14). Despite its constitutive nature, however, eNOS activity can be significantly modified by factors that increase [Ca2+]i (14) or active kinases, particularly Akt, which cause serine phosphorylation of eNOS (5, 7). The latter process appears to be the specific means whereby the mechanostimulus of flow or wall shear stress increases eNOS activity; indeed, some (11, 13) have argued that the level of constitutive eNOS activity is flow rate dependent. Our observations indicate that flow rate is an important but not the sole determinant of basal eNOS activity. Thus blockade of endogenous NO synthesis with L-NMMA significantly reduced the diameter of 1-day-old TMA even in the absence of intravascular flow or pressure. Furthermore, BQ-788 administration caused significant reduction in TMA diameter under both no-flow and flow conditions, indicating that ET-1 activation of the ETB receptor was consistently present and that ETB-induced NO production was independent of that generated by the mechanostimulus of flow. Blockade of SP NK1 receptors was carried out based on our (17) prior observations in blood-perfused intestinal gut loops, where it was demonstrated that SP might participate in the mediation of flow-induced dilation. Observations made in 1-day-old TMA support this contention and are consistent with studies (25) carried out in other circulations. Although endothelial production of SP has been described previously (23), the role of this peptide in intestinal vasoregulation has been generally ascribed to its function as a neurotransmitter within the intrinsic gut nervous system and in sensory afferents (1, 9).

Observations made in these experiments support our contention that NO plays an age-dependent role in regulation of the postnatal circulation. Blockade of endogenous NO synthesis increases net resistance across blood-perfused intestinal gut loops in younger but not older animals (15); furthermore, the concentration of NO within the effluent of buffer-perfused mesenteric arterial arcades is significantly greater in 1- than in 30-day-old animals and is tightly correlated to flow rate only in the younger group (28). Preliminary studies suggest that thoracic aortic endothelial cells from 1- but not 30-day-old swine generate an oxidant burst in response to a shear stimulus of 15 dyn · cm2 that can be detected by oxidant-sensitive fluoroprobes. This oxidant burst is coupled with an increase in NO production by these cells; indeed, the increase in oxidant and NO production that occurs in response to shear stress is blocked by the antioxidants N-acetyl-L-cysteine or diphenyleneiodonium (26). These observations, in concert with data presented in this study, consistently indicate that NO plays an age-dependent role in setting basal vascular tone in newborn intestine and that specific mechanisms relevant to NO production are present at birth.

Vascular Tone in 40-Day-Old TMA Is Determined by Passive Elastic Characteristics of the Vessel

The difference in the hemodynamic behavior of 1- and 40-day-old TMA was striking. Vessels from older swine failed to demonstrate a significant myogenic response to pressure elevation, despite the fact that they exhibit spontaneous tone, i.e., a modest reduction in diameter, when first pressurized to a level consistent with that present in situ. This pattern has been previously described (31) in other circulations. Also, prazosin, losartan, indomethacin, and BQ-610 did not have any significant effect on 40-day-old TMA. The receptors and ion channels necessary to respond to contractile stimuli are almost certainly present in these older vessels, as evidenced by the response of in vivo or in vitro blood-perfused gut loops from 30-day-old animals to exogenous alpha 1-agonists ET-1 and ANG II. It might be argued that resting tone in older TMA might be more dependent on extrinsic neural input than on the first postnatal day inasmuch as Buckley et al. (2) and Gootman et al. (8) have demonstrated a progressive maturation of adrenergic innervation of the swine intestinal vasculature during the first several postnatal months. However, acute denervation (18) or pharmacological blockade of alpha 1-receptors (22) does not significantly alter intestinal vascular resistance in 30- to 40-day-old swine in vivo, and the baseline resistance across denervated, reservoir-perfused and innervated, and autoperfused gut loops is similar at this postnatal age (19, 21). Nor can the relatively relaxed state of 40-day-old TMA be ascribed to the presence of active dilator mechanisms, as neither blockade of endogenous NO synthesis with L-NMMA nor prostacyclin synthesis with indomethacin significantly altered vessel diameter. These observations are consistent with those previously reported (15) using blood-perfused gut loops; thus, unlike the newborn, vascular resistance across 40-day-old intestine is not contingent on constitutive NO production. The lack of significant myogenic response noted in 40-day-old TMA is in contradiction to that reported by Crissinger et al. (4), who demonstrated vasodilation in 1-day-old swine but myogenic vasoconstriction in 3-day and older swine in response to a rapid elevation in venous pressure in vivo. This disparity between our presented in vitro data and the previously reported (4) in vivo data lends further credence to the idea that regulation of the intestinal vasculature may occur at sites other than the TMA. One basis for the unique hemodynamic behaviors of 1- and 40-day-old TMA might be the significant difference in the resting diameters of these vessels. Under in situ conditions, the average TMA diameter is 180 µm on postnatal days 1-3 and 300 µm on postnatal days 32-40 (27). The reactivity of small arteries and arterioles is generally greater as luminal diameter decreases, particularly regarding the efficacy of the myogenic mechanism. In this context, we speculate that 40-day-old TMA function less as resistance vessels and more as simple conduit vessels.

The hemodynamic characteristics of swine TMA significantly change during the first postnatal month. On day 1, intrinsic basal tone is generated by a myogenic mechanism as well as by the constitutive presence of ET-1. This tone is offset, however, by NO, whose basal production is affected by the mechanostimulus of flow and also by constitutive activation of ETB and NK1 receptors. In contrast, on postnatal day 40, TMA diameter is essentially determined by the passive elastic characteristics of the vessel, as there is no evidence of active constrictor or dilator tone under resting conditions.


    ACKNOWLEDGEMENTS

We thank Karen Watkins for providing outstanding secretarial support to our laboratory.


    FOOTNOTES

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

Address for reprint requests and other correspondence: C. A. Nankervis, Children's Hospital, 700 Children's Dr., Columbus, OH 43205 (E-mail: nankervisc{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 5 June 2000; accepted in final form 14 November 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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