Endothelin ETA and ETB receptors in postnatal intestine

Craig A. Nankervis1, David J. Dunaway2, and Charles E. Miller2

1 Department of Pediatrics, College of Medicine and Public Health, The Ohio State University and 2 Children's Research Institute, Children's Hospital, Columbus, Ohio 43205


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We aimed to characterize endothelin (ET) receptors in the swine intestinal vasculature and to determine ischemia-reperfusion (I/R) effects on these receptors. Saturation and competitive binding assays were performed on mesenteric artery protein membranes from 1- and 40-day-old animals, both control and those subjected to 1 h of partial ischemia followed by 6 h of reperfusion in vivo. Scatchard analysis of saturation binding with 125I-labeled ET-1 in membranes from endothelium-denuded (E-) vessels revealed that the maximum number of binding sites was greater in younger animals. Competitive 125I-ET-1 binding was significant for a one-site model with ET-1, ET-3, and sarafotoxin S6c (S6c) in membranes from endothelium-intact (E+) and E- vessels in both age groups. The maximum number of ET-1 binding sites was significantly greater in younger animals. In the presence of the ETA receptor antagonist BQ-123, competitive 125I-ET-1 binding was significant for a one-site model with ET-1 and S6c in membranes from E+ vessels in both age groups. The maximum number of ET-1 binding sites was significantly greater in younger animals. After I/R, the maximum number of ET-1 binding sites was unchanged. In the presence of BQ-123, specific binding by ET-1 and S6c was eliminated in both age groups after I/R. These results suggest that both ET receptor populations are expressed to a greater degree in younger animals and I/R significantly affects the ETB receptor.

swine; newborn intestine; intestinal circulation physiology


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

A POTENT VASOACTIVE 21-amino-acid peptide, endothelin-1 (ET-1) was first isolated by Yanagisawa et al. (27) from the conditioned medium of cultured porcine aortic endothelial cells. Three isoforms of ET have been described: ET-1, ET-2, and ET-3. ET-1 exerts its effects through the activation of at least two distinct receptor subtypes designated ETA and ETB (1, 9, 18, 23). These seven-transmembrane-spanning, G protein-coupled receptors share similar signal transduction pathways. Thus receptor activation increases intracellular Ca2+ concentration via phospholipase C-mediated production of inositol triphosphate and diacylglycerol (21, 22). The ETA receptor, located on vascular smooth muscle (VSM), is selective for ET-1 and ET-2, and its activation leads to vasoconstriction (2, 3). The ETB receptor, located on the endothelium and VSM, has equal affinity for all three isopeptides. Activation of the endothelial ETB receptor leads to vasodilation mediated via nitric oxide (NO) production and release (4, 20, 23). Activation of the VSM ETB receptor causes vasoconstriction.

The role of ET-1 in regulation of swine intestinal hemodynamics and oxygenation changes during early postnatal life. For example, blockade of ETA receptors increases intestinal oxygen extraction and the capillary filtration coefficient in 3- but not 35-day-old animals, indicating that ET-1 exerts a tonic constrictive effect on precapillary sphincters and hence capillary perfusion in an age-specific fashion (12). Also, although the dose-response curve for ET-1 in mesenteric artery rings is similar in 3- and 35-day-old animals, pretreatment with the selective ETB antagonist BQ-788 significantly shifts the curve leftward in 3- but not 35-day old animals (13). Finally, sarafotoxin S6c (S6c), a snake venom peptide that has significant sequence homology to the ET isopeptide family and binds exclusively to the ETB receptor, elicits significantly greater vasodilation in precontracted mesenteric artery rings from younger rather than older animals (13). Collectively, these observations suggest that the binding affinity or receptor number of the ETA and ETB receptors within the intestinal circulation may be developmentally regulated during early postnatal life.

Further support of this possibility is derived from the response of 3- and 35-day-old intestine to ischemia-reperfusion (I/R). Younger animals demonstrate a sustained vasoconstriction after I/R that can be significantly attenuated by prior blockade of ETA receptors; in contrast, older animals display a sustained vasodilation under the same circumstance (13). Also, mesenteric artery rings exposed to I/R in vivo demonstrate a significant leftward shift in the ET-1 dose-response curve, reminiscent of the effect of selective ETB receptor blockade and, once again, this effect is only present in rings from 3-day-old animals (13). One interpretation of these observations is that I/R causes a selective ablation of ETB receptors in younger intestine.

Based on these observations, we hypothesized that the number of ETA and ETB receptors are developmentally regulated; specifically, newborn intestine has a greater number of these receptors, particularly the endothelial ETB subtype. To test this hypothesis, saturation and competitive binding assays were carried out on membrane prepared from the mesenteric arteries of 1- and 40-day-old swine, under both control and post-I/R conditions.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Care and handling of experimental animals. Studies were conducted using farm-bred swine of either sex. Two age groups were studied: 1 day old (newborn) and 40 day old (weanling). All animals were obtained on the day before use and fasted for 12-18 h before surgery. At the end of the first postnatal month, swine had been removed from the sow, weaned from milk, and placed on a grain-based diet. Although not an adult, the 40-day-old weanling piglet has undergone significant postnatal maturation, particularly from the standpoint of intestinal function (5, 19, 25). Surgery was carried out under anesthesia with tiletamine HCl-zolazepam HCl (7.5 mg/kg im) and xylazine (5 mg/kg im) and maintained with pentobarbital sodium (5 mg · kg-1 · h-1 iv); the animals were killed by overdose with pentobarbital sodium (6 mg/kg iv). 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]. All experimental protocols were approved and monitored by the Institutional Animal Care and Use Committee of the Children's Research Institute. Animals from each age group were divided into two subgroups. Animals assigned to the experimental group were exposed to I/R in vivo. Control animals underwent an identical surgical procedure with the exception of creation of the mesenteric artery coarctation. The mesenteric artery from each animal was used in the binding assays.

Induction of I/R in vivo. Each animal was anesthetized, an airway was established via a midline tracheotomy, and ventilation was started and adjusted to maintain normal blood gas tensions. The mesenteric artery was isolated by retroperitoneal dissection. In animals undergoing I/R, a mesenteric artery coarctation was created by placing a 3-0 silk ligature around the superior mesenteric artery at its origin from the abdominal aorta. A small piece of vinyl tubing (V5 for 3-day-old animals, V7 for 35-day-old animals; Bolab, Lake Havasu City, AZ) was placed in between the artery and the ligature before tightening. After tightening, the vinyl tubing was removed. This action created a precise degree of coarctation that reduced mesenteric blood flow by ~75% in both age groups (13). The ligature was removed after 1 h and reperfusion allowed for 6 h. Non-I/R animals were treated in an identical way except that the ligature was not tightened.

Membrane preparation. The entire mesenteric artery (E+) was removed en bloc, snap frozen in liquid nitrogen, and stored at -80°C until use. In some cases, before freezing, the vessel was opened longitudinally and the endothelial layer removed by gentle scraping of the luminal surface (E-). Vessel segments were pulverized to a fine powder within a steel chamber while still frozen at -80°C. In all cases, great care was taken to keep the membrane preparation cold by performing all the following steps on ice. Powdered vessels were suspended in homogenization buffer (50 mM Tris · HCl, pH 7.4, 5 mM MgCl2, 5 mM EDTA, and 1 mM EGTA) and homogenized with a polytron at maximal setting on ice. Homogenates were centrifuged at 750 g for 5 min at 4°C between each step, and membrane-containing supernatants were saved and combined for a final centrifugation at 20,000 rpm for 35 min at 4°C to pellet the membranes. Membrane pellets were reconstituted in assay buffer (50 mM HEPES and 5 mM MgCl2) via tituration through a progressive series of 18-, 21-, 23-, and 25-gauge needles. An aliquot of the final membrane preparation was used to assay for total protein with the bicinchoninic acid protein assay (Pierce, Rockford, IL).

Saturation and competition binding assays. A series of preliminary experiments was performed to optimize binding conditions for 125I-labeled-ET-1. To determine a suitable membrane concentration, increasing amounts of membrane (10-100 µg) from E- vessels were incubated with 125I-ET-1. From these data, 50 µg of protein per sample were chosen for the assays as this concentration was on the linear portion of the membrane concentration vs. maximal 125I-ET-1 binding curve (data not shown). An incubation time of 2 h at 23°C was selected for the assays because 125I-ET-1 binding reached equilibrium under these conditions. All binding experiments were carried out in a total volume of 0.5 ml assay buffer (50 mM HEPES, 5 mM MgCl2, and 0.3% BSA).

Total 125I-ET-1 binding was determined in saturation studies by incubating membrane samples (50 µg) prepared from E- vessels with increasing concentrations of 125I-ET-1 (12.5-200 pmol/l). Nonspecific binding was determined in parallel incubations containing excess unlabeled ET-1 (1 µM). After the 2-h incubation period, the assay was halted by the addition of 0.5 ml ice-cold assay buffer to the reaction mixture. Bound and free ligand were separated via centrifugation (1,500 rpm at 4°C for 10 min) followed by aspiration of the free ligand containing supernatant. Pellets were washed once with 1 ml ice-cold 50 mM Tris · HCl. Final bound ligand containing membrane pellets were counted for 125I for 2 min in a gamma counter (model 5010, Packard Instrument, Meriden, CT). Specific binding was calculated as the difference between total and nonspecific binding. Nonspecific binding was consistently <20% of total binding for each separate assay.

Competitive binding experiments were performed in triplicate with membrane (50 µg) prepared from E+ or E- vessels and 125I-ET-1 (33.3 pmol/l) plus decreasing concentrations of unlabeled ET-1 (10-6-10-11 M), ET-3 (10-6-10-11 M), and S6c (10-6-10-11 M) as competitor. S6c is a snake venom peptide with significant sequence homology to the ET isopeptide family; it binds exclusively to the ETB receptor and is therefore a useful ligand to discriminate between ETA and ETB receptors (26). Nonspecific binding was determined by the addition of 1 µM unlabeled competitor. Specific binding was calculated as the difference between total binding and nonspecific binding.

In some cases, pharmacological blockade of the ETA receptor was performed to eliminate ETA binding before carrying out the binding assay to determine the presence of the endothelial ETB receptor. To this end, the nonpeptide ETA receptor antagonist BQ-123 (5 × 10-5 M) was added to membrane prepared from E+ and E- vessels and allowed to incubate for 1 h before carrying out competitive binding assays with decreasing concentrations of unlabeled ET-1 (10-6-10-11 M) and S6c (10-6-10-11 M) as competitive ligands (7). The amount of BQ-123 added was five orders of magnitude greater than the dissociation constant (Kd) for the ETA receptor established in preliminary studies (i.e., ETA Kd ~0.25 nM). After the 2-h incubation period at 23°C, the assay was halted by the addition of 0.5 ml ice-cold assay buffer to the reaction mixture. Bound and free ligand were separated via centrifugation (1,500 rpm at 4°C for 10 min) followed by aspiration of the free ligand containing supernatant. Pellets were washed once with 1 ml ice-cold 50 mM Tris · HCl. Final bound ligand containing membrane pellets were counted for 125I for 2 min in a gamma counter (model 5010, Packard Instrument). Specific binding was calculated as the difference between total binding and nonspecific binding. Nonspecific binding was typically 10-20%.

Data analysis. All observations were made in triplicate, and the average response was determined. In statistical analysis and data presentation, n refers to the number of animals in which observations were made. Data are expressed as means ± SE. Concentrations expressed represent the final concentration in the reaction mixture. Kd (in nM) and Bmax (maximum number of binding sites, in fmol/mg protein) using ET-1 as the competitive ligand were calculated for each membrane sample using the nonlinear curve-fitting RADLIG computer program (11). Single and multiple site models were statistically compared to determine the best fit by comparing the residual variance using an F test. The model that yielded the greatest degree of significance was chosen. Responses were compared between age groups by a three-way ANOVA, which utilized group (1 vs. 40 day old), condition (control vs. I/R), and drug concentration as the main effects. Post hoc Tukey B tests were carried out on data sets in which the F statistic was significant (P < 0.01), to determine the sites of significant differences between variables.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Binding studies carried out in membrane prepared from control animals. Specific binding of 125I-ET-1 to membranes prepared from E- mesenteric arteries was a saturable process in membranes prepared from both age groups (Fig. 1). Scatchard analysis of the saturation binding curves generated similar Kd values of 0.24 ± 0.05 and 0.16 ± 0.07 nM for 1- and 40-day-old subjects, respectively. The Bmax was significantly higher in 1- vs. 40-day-old animals (277 ± 104 vs. 29 ± 13 fmol/mg protein, respectively, P < 0.01).


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Fig. 1.   Saturation binding curves for mesenteric artery with no endothelial layer (E-) from 1- and 40-day-old control animals. Top: saturable specific binding of 125I-labeled endothelin-1 (ET-1) for the 1-day-old group. Bottom: saturable specific binding of 125I-ET-1 for the 40-day-old group. , , Total binding; open circle , , specific binding; triangle , black-triangle, nonspecific binding. Membranes were incubated with increasing concentrations of 125I-ET-1 for 2 h at 23°C. Nonspecific and total binding was determined in the presence and absence of 1 µM unlabeled ET-1, respectively. Specific binding was defined as the difference between total and nonspecific binding. The data shown is a representative saturation binding curve for each age group from 1 of 4 experiments, each performed in triplicate. The apparent dissociation constant (Kd) values estimated from Scatchard analysis of the mean saturation data were 0.24 ± 0.05 and 0.16 ± 0.07 nM and the maximum number of binding sites (Bmax) values were 277 ± 104 and 29 ± 13 fmol/mg protein in 1- and 40-day-old swine, respectively.

Membranes prepared from E- vessels from both age groups demonstrated competition binding data that were best fit by a one-site model (Fig. 2). The efficacy of displacement of 125I-ET-1 was ET-1 > ET-3 S6c. This profile is consistent with the isopeptide-selective ETA receptor. The Kd values generated using ET-1 as the competitive ligand were similar in both age groups; however, the Bmax values were significantly greater in membrane prepared from younger animals (Table 1). These observations identify the presence of ETA receptors on VSM and further indicate that the number of receptors is significantly greater in membrane prepared from younger animals.


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Fig. 2.   Competition binding curves for E- mesenteric artery from 1- and 40-day-old control animals. Top: 1-day-old group. Bottom: 40-day-old group. open circle , , Competition with ET-1; triangle , black-triangle, competition with ET-3; , , competition with sarafotoxin S6c (S6c). The competitive binding profile is consistent with the isopeptide-selective ETA receptor, and the absence of the endothelium localizes these ETA receptors to the vascular smooth muscle. The Kd and Bmax values for these binding curves are presented in Table 1. Data are given as means ± SE; n = 8 for all observations.


                              
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Table 1.   Kd and Bmax for ET-1 in membranes from E- mesenteric arteries from 1- and 40-day-old control animals

A similar pattern emerged in the competitive binding assays carried out on E+ vessels. Thus membranes prepared from E+ vessels from both age groups demonstrated competition binding data that were best fit by a one-site model (Fig. 3). The efficacy of displacement of 125I-ET-1 was ET-1 > ET-3 S6c, once again consistent with the isopeptide-selective ETA receptor. The Kd values generated using ET-1 as the competitive ligand were similar in both age groups and were also statistically similar to values obtained in E- vessels. The Bmax values were significantly greater in membrane prepared from younger animals and were also statistically similar to values obtained in E- vessels (Table 2).


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Fig. 3.   Competition binding curves for entire mesenteric artery (E+) from 1- and 40-day-old control animals. Top: 1-day-old group. Bottom: 40-day-old group. Symbols same as given in Fig. 2 legend. The competitive binding profile is consistent with the isopeptide-selective ETA receptor. The Kd and Bmax values for these binding curves are presented in Table 2. Data are given as means ± SE; n = 3 for all observations.


                              
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Table 2.   Kd and Bmax for ET-1 and receptor subtype distribution in membranes from E+ mesenteric arteries from 1- and 40-day-old control BQ-123-treated animals

To delineate the presence of ETB receptors, E+ membranes were first treated with the selective ETA antagonist BQ-123. The concentration of BQ-123 used for blockade (5 × 10-5 M) was five orders of magnitude greater than the Kd for the ETA receptor, assuring a complete blockade of the ETA receptor population. This blockade reduced total specific binding of E+ membranes by 93% in 40-day-old animals, but only by 79% in 1-day-old animals, indicating that the remaining population of ET receptors was greater in younger animals (Table 2). Competitive binding of this residual receptor population revealed an efficacy of displacement of 125I-ET-1 that was equal for ET-1 and S6c (Fig. 4). This binding profile is consistent with the ETB receptor, which is nonisopeptide selective. The binding data were best fit by a one-site model, and the Kd values generated with ET-1 as the competitive ligand were similar in both age groups (Table 2). However, the Bmax values indicated that the number of ETB receptors was significantly greater in membrane prepared from younger animals, consistent with the effect of BQ-123 on total specific binding. This assay was repeated in membrane prepared from E- vessels from both age groups. Neither group demonstrated specific competitive binding after blockade of the ETA receptors with BQ-123. Collectively, these observations indicate the presence of ETB receptors on the endothelium, but not VSM; furthermore, the number of ETB receptors is significantly greater in membrane prepared from younger animals.


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Fig. 4.   Competition binding curves for E+ mesenteric artery from 1- and 40-day-old control animals after blockade of the ETA receptor population with BQ-123. An excess of BQ-123 (5 × 10-5 M) was added to the membrane preparation before carrying out the competitive binding assay. Top: 1-day-old group. Bottom: 40-day-old group. open circle , , Competition with ET-1; , , competition with S6c. Note that ET-1 and S6c provide equal competition, identifying the residual (i.e., post-BQ-123 blockade) receptor population as the nonisopeptide-selective ETB receptor. The Kd and Bmax data for these curves are presented in Table 2. Data are given as means ± SE; n = 3 for all observations.

Binding studies carried out in membrane prepared from animals exposed to intestinal I/R in vivo. Membrane prepared from E+ vessels exposed to I/R in vivo demonstrated competitive binding profiles similar to those noted under control conditions in both age groups. Thus both age groups demonstrated binding data that were best fit by a one-site model with an efficacy of displacement of 125I-ET-1 that was ET-1 S6c, a pattern consistent with the ETA receptor (Fig. 5). The Kd values generated using ET-1 as the competitive ligand were similar in both age groups and were also similar to the values obtained in E+ control vessels. Similar to the E+ control vessels, the Bmax values were significantly greater in younger animals (Table 3). These observations indicate that the population of ETA receptors was not affected by the perturbation of I/R in either age group.


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Fig. 5.   Competition binding curves for E+ mesenteric artery from 1- and 40-day-old animals exposed to ischemia-reperfusion (I/R) in vivo. Top: 1-day-old group. Bottom: 40-day-old group. Symbols same as given in Fig. 4 legend. Note that the competition is greater for ET-1 than for S6c, a pattern consistent with the isopeptide-selective ETA receptor. The Kd and Bmax data for these curves are presented in Table 3. Data are given as means ± SE; n = 3 for all observations.


                              
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Table 3.   Kd and Bmax for ET-1 in membranes from E+ mesenteric arteries from 1- and 40-day-old animals after I/R

Blockade of the ETA receptor population with BQ-123 was also carried out in membrane prepared from mesenteric artery exposed to I/R in vivo. In younger animals, specific competitive binding with ET-1 or S6c was eliminated by in vivo exposure of the mesenteric artery to I/R (Figs. 6 and 7). The RADLIG program would not generate Kd or Bmax values for these binding data because of the lack of specific binding for ET-1. These observations indicate that the endothelial ETB population was essentially eliminated by the perturbation of I/R in mesenteric artery from 1-day-old animals. In contrast to the younger age group, membrane prepared from mesenteric artery from 40-day-old animals exposed to intestinal I/R demonstrated competitive binding data similar to that noted in control animals (Figs. 6 and 7).


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Fig. 6.   Competition binding curves for 1-day-old E+ mesenteric artery exposed to I/R in vivo in membrane pretreated with BQ-123. One-day-old animals were exposed to I/R in vivo; thereafter, membrane was prepared for binding assays. BQ-123 was administered to the membrane before carrying out the binding assay to block the ETA receptors, so that the competitive binding data shown here represents the residual ETB receptor population. Top: curves generated using ET-1 (open circle ) as the competitive ligand. Bottom: curves generated using S6c () as the competitive ligand. Top and bottom: control data are shown for comparison with post-I/R data (diamond ); these control data are identical to those presented in Fig. 4. Note the loss of specific competitive binding in membrane prepared from mesenteric artery exposed to I/R in vivo. The RADLIG program was unable to generate Kd and Bmax data from the I/R binding data. These observations indicate a substantial loss of the ETB receptor population after I/R. Data are given as means ± SE; n = 3 for all observations.



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Fig. 7.   Competition binding curves for 40-day-old E+ mesenteric artery exposed to I/R in vivo in membrane pretreated with BQ-123. Forty-day-old animals were exposed to I/R in vivo; thereafter, membrane was prepared for binding assays. BQ-123 was administered to the membrane before carrying out the binding assay to block the ETA receptors, so that the competitive binding data shown here represents the residual ETB receptor population. Top: curves generated using ET-1 () as the competitive ligand. Bottom: curves generated using S6c () as the competitive ligand. Top and bottom: control data are shown for comparison with post-I/R data (black-lozenge ); these control data are identical to those presented in Fig. 4. Data are given as means ± SE; n = 3 for all observations.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Data from these experiments support the working hypothesis, i.e., the presence of ETA and ETB receptors within the postnatal intestinal circulation is developmentally regulated. Both age groups demonstrated a receptor whose ligand binding profile was consistent with the ETA receptor, and these receptors were located on VSM; however, the number of VSM ETA receptors was significantly greater in mesenteric artery harvested from 1-than 40-day-old animals (1-3, 9). Blockade of the ETA receptor population with BQ-123 uncovered a second receptor whose ligand profile identified it as the ETB receptor (18, 20). This receptor was localized to the endothelium in both age groups; however, older animals demonstrated a marked paucity of the ETB receptor compared with the 1-day-old group. Thus mesenteric artery from 1-day-old animals contains VSM-based ETA receptors and, especially, endothelial-based ETB receptors in a substantially greater quantity than from 40-day-old animals.

These receptor binding data correlate well with our (12, 13) prior observations regarding the hemodynamic effects of ET-1 in the postnatal intestinal circulation. For example, the contractile response of mesenteric artery rings to exogenous ET-1 is similar in both age groups; however, only rings from younger animals demonstrate a significant leftward shift of the ET-1 dose-response curve after selective blockade of the ETB receptors with BQ-788 or mechanical removal of the endothelium. Similarly, administration of exogenous ET-1 to in vivo gut loops leads to a greater rise in intestinal vascular resistance in younger but not older animals after pretreatment with BQ-788 (8). Also, S6c, a selective ETB receptor agonist (26), causes a far greater degree of dilation in precontracted mesenteric artery rings from younger animals. These hemodynamic effects are entirely consistent with the binding data presented in this report; thus a large population of endothelium-based, dilatory ETB receptors would counterbalance the constrictor effects generated by the ETA receptor. Activation of the ETB receptor leads to a brisk rise in intracellular Ca2+ concentration within the endothelial cell via the phospholipase C-inositol triphosphate-diacylglycerol signal transduction pathway (21, 22), which in turn stimulates the endothelial isoform of NO synthase (4, 20, 23). Activation of endothelial ETB receptors would therefore generate the dilatory agent NO. It is interesting that the significantly greater number of ETA receptors in 1-day-old intestine is neatly counterbalanced by a far greater population of ETB receptors at this postnatal age, i.e., that developmental regulation of both receptors occurs so as to maintain a relative hemodynamic balance of the constrictor and dilator actions of ET-1.

Why are there so many ETA receptors present in 1-day-old mesenteric artery? It is not logical to expect that their presence relates to resistance regulation for at least two reasons. First, intestinal vascular resistance is significantly lower in 1- than 40-day-old swine intestine, not higher as one might expect in the presence of an exceptionally large number of contractile ETA receptors (15). Second, blockade of ETA receptors with BQ-610 under in vivo conditions has little effect on intestinal vascular resistance when hemodynamic conditions are normal (12). However, the role of the ETA receptor significantly transcends that of hemodynamic regulation; indeed, one of its primary functions is to facilitate angiogenesis and vascular remodeling (6, 16, 17). We speculate that the abundance of ET receptors in newborn intestine reflects this angiogenic role. The intestinal mass increases dramatically in swine during the first postnatal month with respect to overall body growth (25); thus the intestinal mass is ~80 g in a total body wt of ~1.5 kg on the first postnatal day (ratio: 0.053) and 600 g in a total body wt of 4 kg by the end of the first postnatal month (ratio: 0.15). This rapid rate of growth necessitates the generation of new vasculature. The substantial presence of endothelial ETB receptors permits the existence of relatively large amounts of ET-1 within the newborn intestine (12) without causing overwhelming vasoconstriction.

The data from these experiments also permit a more detailed interpretation of the age-dependent hemodynamic effects of I/R previously reported by Nowicki (14) from this laboratory. Partial ischemia for 1 h followed by 2 h of reperfusion leads to a sustained vasoconstriction in newborn animals (14). Here we observed a selective loss of the endothelial ETB receptor population in 1-day-old mesenteric artery after a similar I/R insult, whereas the ETA receptor population was unaffected. Loss of the dilatory ETB receptor population would upset the balance normally present between the ETA and ETB receptors, favoring constriction. These events at the receptor level would explain the development of sustained constriction after I/R in newborn intestine. In this context, Luscher (10) and Vanhoutte and Shimokawa (24) have demonstrated that the perturbation of I/R within the coronary circulation selectively attenuates endothelial cell function, leaving VSM function intact and causing an imbalance between locally generated constrictor and dilator forces. These reports are not specific for ET receptors, but do highlight a similar pattern demonstrated (14) in the newborn intestinal circulation, i.e., selective I/R-induced damage to the endothelium, leading to a relative loss of endothelium-derived dilatory forces.

We conclude that the ET receptor population in postnatal mesenteric artery is age dependent, thus 1-day-old animals have a significantly greater population of ETA and ETB receptors than do older animals. Furthermore, we conclude that the effect of partial I/R on the ET receptor population is receptor dependent, thus the ETB receptor population is selectively ablated by partial I/R whereas the ETA receptor is relatively unaffected.


    ACKNOWLEDGEMENTS

We thank Dr. Philip Nowicki for critical review of the manuscript and Karen Watkins for secretarial support.


    FOOTNOTES

This study was supported by Children's Research Institute Grant 213798.

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 30 June 2000; accepted in final form 24 October 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastrointest Liver Physiol 280(4):G555-G562
0193-1857/01 $5.00 Copyright © 2001 the American Physiological Society




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