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
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ABSTRACT |
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
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INTRODUCTION |
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.
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METHODS |
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.
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RESULTS |
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; , , specific binding;
, , 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.
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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. ,
, Competition with ET-1; ,
, 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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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
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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. ,
, 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.
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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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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 ( ) 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
( ); 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
( ); these control data are identical to those presented
in Fig. 4. Data are given as means ± SE; n = 3 for all observations.
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DISCUSSION |
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.
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ACKNOWLEDGEMENTS |
We thank Dr. Philip Nowicki for critical review of the manuscript
and Karen Watkins for secretarial support.
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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.
 |
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