(Received for publication, August 16, 1994; and in revised form, October 18, 1994)
From the
The binding of I-neurotensin (NT) to human
umbilical vein endothelial cell monolayers was studied. At 20 °C,
I-NT bound to a single class of binding sites with a
dissociation constant of 0.23 ± 0.08 nM and a binding
site density of 5500 ± 1300 sites/cell (n = 3).
I-NT also bound to human aortic endothelial cells with a
dissociation constant of 0.6 ± 0.26 nM and a binding
site density of 32000 ± 1700 sites/cell. Association and
dissociation kinetics were of a pseudo-first order and gave association
and dissociation rate constant values of 1.6
10
M
s
and 3.5
10
s
, respectively.
I-NT binding was inhibited by NT analogues with a rank
order of potency similar to that characterizing brain high affinity NT
binding sites (K
, nM):
NT
(0.11) > NT (0.35) >
acetyl-NT
(1.5) >
[Phe
]NT (12) >
[D-Tyr
]NT (>1000).
I-NT binding was also inhibited by the non-peptide NT
antagonist SR 48692 (K
= 16
nM) but was not affected by levocabastine, an inhibitor of low
affinity brain NT binding sites. NT had no effect on cGMP levels in
endothelial cells but NT and its analogues increased
Ca
efflux from endothelial cells at
nanomolar concentrations with a rank order of potency which was
identical to that observed in binding experiments. This effect was
inhibited by SR 48692 (IC
= 8 nM). NT was
able to increase phosphoinositide turnover in these cells, and this
effect was blocked by SR 48692. The correlation between dissociation
constants of NT analogues in binding experiments and IC
values in
Ca
efflux experiments was very
high (r = 0.997) with a slope near unity, indicating
that
I-NT binding sites are functional NT receptors
coupled to phosphoinositide hydrolysis and Ca
release
in human umbilical vein endothelial cells.
The tridecapeptide neurotensin (NT) ()(1) has been shown to act as a neurotransmitter in
the central nervous system, and it has been suggested that it acts as a
local hormone in peripheral tissues(2) . Peripheral NT is
localized essentially in the endocrine N-cells of intestinal
mucosa(3, 4) . Plasma NT levels in humans which
increase after food intake (2) may modulate peristaltic
intestinal contractions(5) . Depending on the location, NT is
able to contract or relax intestinal muscle either directly through
interaction with smooth muscle cells or indirectly through the
activation of neurotransmitter release from intramural nerve
endings(2) . These conclusions are based on indirect evidence
based on the blockade of neurotransmitter release by tetrodotoxin.
However, binding studies using iodinated NT
derivatives(6, 7, 8) , as well as
electrophysiological experiments on isolated smooth muscle
cells(9) , provide convincing evidence that some effects of NT
are due to direct interaction of the peptide with intestinal smooth
muscle cell receptors.
In contrast to the intestinal effects of NT, the mechanisms of the cardiovascular effects of this peptide are much less well characterized, and it is still unclear if NT acts directly or indirectly on vascular cells. Indeed, intravenous infusion of NT produces species-dependent systemic as well as regional cardiovascular effects(1, 10, 11, 12, 13) . The effects of NT have also been studied in isolated organ experiments, where NT contracted rat portal vein by a histamine-independent mechanism(14) , but had no contractile activity on rabbit pulmonary artery(15) , rat aorta(16) , or dog carotid arteries(17) . In these latter experiments, an endothelium-dependent relaxing effect could be demonstrated(17) , suggesting that NT may induce smooth muscle relaxation through a direct interaction with endothelial cells via an activation of NO synthase. Subcutaneous injection of NT also produces an increase in cutaneous vascular permeability which can be attributed to either a direct effect of NT on vascular endothelial cells or an indirect histamine-related mechanism (18) .
Despite these numerous in vivo and in vitro pharmacological studies, no NT binding sites have been described on vascular smooth muscle or endothelial cells yet. NT binding to mast cells has been reported(19, 20) , but the low affinity and specificity of these binding sites raise several questions concerning their potential implication in histamine release(2) . We hereby describe the existence and characterization of high affinity NT receptors on human umbilical vein and aortic endothelial cells, suggesting that some of the cardiovascular effects of NT might be due to direct interaction of NT with endothelial cells.
where Q = quantity of
Ca
lost from the cells during time
period i and Q
= quantity of
Ca
in the cells at the end of the
experiment.
Ca
efflux was stimulated by
addition of NT to the washing solution after eight medium changes. When
the effects of antagonists were studied, they were present from the
beginning of the experiment, i.e. for 4 min before the
addition of NT. For concentration-effect relationships and inhibition
experiments,
Ca
efflux during the 1st
min of stimulation was calculated by summation and corrected by
subtracting base-line efflux. Data from several experiments were pooled
and analyzed together by fitting the sigmoidal equation to the data by
nonlinear regression thus determining EC
values and their
standard errors (23) using the program Sigmaplot (Jandel
Scientific, Erkrath, Germany).
As shown in Fig. 1A, I-NT
binding to HUVEC monolayers at 20 °C was saturable, with a
nonspecific binding representing less than 20% of total binding at all
concentrations. The Scatchard plot (Fig. 1A, inset) was linear, indicative of a single class of
non-interacting binding sites. From several experiments, the mean
dissociation constant (K
) was found to be of 0.23
± 0.08 nM and the binding site density (B
) represented 5500 ± 1300 sites/cell (n = 3). Binding studies carried out at 37 °C gave
results which were identical to those from experiments performed at 20
°C, with K
= 0.40 ± 0.23 nM and B
= 5800 ± 1500
sites/cell (n = 2). Further experiments were therefore
carried out at 20 °C in order to study binding at NT affinities
close to the physiological range while minimizing internalization and
degradation of NT which is likely to occur at 37 °C. At this
temperature, NT binding sites could also be detected in HAECs, where K
= 0.6 ± 0.26 nM and B
= 32,000 ± 1,700 sites/cell (n = 3, not shown).
Figure 1:
I-NT
binding to HUVEC monolayers. A, equilibrium binding
experiments. Confluent cells were incubated for 1 h at 20 °C with
increasing concentrations of
I-NT in the presence or
absence of 1 µM of unlabeled NT. The specific binding
(
) was determined as the difference between total (
) and
nonspecific binding (
). Results are from one experiment
representative of a total of three experiments performed in triplicate. Inset, Scatchard plot of the specific binding, for this
particular experiment K
= 0.12
nM and B
= 3600 sites/cell. B, association kinetics of
I-NT binding.
Confluent cells were incubated at 20 °C with
I-NT
(100 pM) and the reaction was terminated at different time
points by two rapid washes with ice-cold PSS. Nonspecific binding was
determined in the presence of 1 µM of unlabeled NT and was
substracted from total binding to give specific binding at each time
point. Data are the mean of four experiments performed in triplicate.
The solid line represents the fit of a monoexponential
function to the data, allowing to determine k
. C, dissociation of
I-NT binding to HUVECs. After
1 h of incubation with 100 pM
I-NT, 1 µM of unlabeled NT (
) or of SR 48692 (
) was added and the
reaction terminated at different time points by two rapid washes with
PSS. Data are the mean of three experiments performed in triplicate.
The solid line represents the fit of a monoexponential
function to the data, which allowed for the calculation of k
.
The kinetics of I-NT binding indicated that the association of
I-NT (100 pM) was complete after 1 h of
incubation and remained stable for up to 2 h. As shown in Fig. 1B, the mean data were well described by a
monoexponential equation corresponding to a pseudo-first order
association reaction, with k
= 0.031
min
(t
= 22 min).
Dissociation of
I-NT could be induced by either unlabeled
NT or SR 48692, a specific non-peptide antagonist of high affinity NT
receptors, and was identical in both cases (Fig. 1C).
Dissociation was rapid, with a half-life of 33 min, corresponding to a
dissociation rate constant (k
) of 3.5
10
s
. A small percentage
of
I-NT (around 20%) did not dissociate and remained
associated to the cells even after 3 h of incubation. The association
rate constant k
was found to be 1.6
10
M
s
.
Calculating the dissociation constant K
= k
/k
gave a value
of 0.22 nM, identical to the K
value
determined in equilibrium binding experiments.
As shown in Fig. 2, NT and its analogues inhibited I-NT
binding in a monophasic manner, slope factors being close to unity. As
the concentration of
I-NT used (100 pM) was less
than half of the K
value of NT, the concentrations
of compounds giving 50% of inhibition of
I-NT binding (K
) can be considered equal within experimental
error to the affinities of the compounds for
I-NT binding
sites. The following rank order of potency was determined (K
± S.E. nM, n = 2-3): NT
(0.11 ± 0.06),
NT (0.35 ± 0.05), acetyl-NT
(1.5 ±
0.7), [Phe
]NT (12 ± 5).
[D-Tyr
]NT was inactive up to 10
µM.
I-NT binding could also be inhibited by
SR 48692, with a K
value of 16 ± 4
nM. The rank order of potency of NT analogues and the
inhibition by SR 48692 show that endothelial cell NT binding sites were
very similar to rat brain NT receptors(21) . In rat and mouse
brain, two different classes of
I-NT binding sites have
been distinguished using the antihistaminergic compound levocabastine
which specifically inhibited
I-NT binding to low affinity
sites(28, 29) . However, in endothelial cell
monolayers levocabastine had no effect on
I-NT binding up
to a concentration of 10 µM (not shown), suggesting that
HUVEC NT receptors were of the high affinity type.
Figure 2:
Effect of NT analogues and SR 48692 on I-NT binding to HUVECs. HUVEC monolayers were incubated
with
I-NT (100 pM) and unlabeled NT (
),
NT
(
), acetyl-NT
(
), [Phe
]NT (
), and SR 48692
(
) for 1 h at 20 °C. Results, expressed as percent of
specific
I-NT binding remaining in the presence of the
compounds, are from one representative experiment performed in
triplicate. The solid lines represent a fit of the sigmoidal
equation to the data.
The effect of
several peptides, which, like NT, release histamine from mast cells,
was assessed on I-NT binding to endothelial cells:
neither substance P, nor bradykinin or other peptides like angiotensin
II, vasoactive intestinal peptide, or corticotropin-releasing factor
had any effect on
I-NT binding to HUVECs at 10 µM (not shown).
The presence of NT receptors in HUVECs was
visualized after RT-PCR amplification of the NT receptor mRNA extracted
from HUVECs which showed only one 269-bp single band after
electrophoresis (Fig. 3A). As shown in Fig. 3B, this band was identified as a NT receptor
mRNA-derived amplicon by Southern blotting, whereas no amplification
products could be detected in the control samples. NT has been shown to
increase phosphoinositide turnover and activate intracellular free
Ca release in HT29 colonic cancer cells (30) and rat NT receptor transfected Chinese hamster ovary
cells(31) , whereas it increased cGMP levels and activated NO
synthesis in N1E-115 neuroblastoma
cells(32, 33, 34) . In HUVECs, we were unable
to demonstrate an increase in cGMP levels induced by NT (3-100
nM), whereas the calcium ionophore A23187 (1 µM)
increased cGMP levels more than 5-fold from 0.28 to 1.6 pmol/10
cells (not shown). However, NT was able to modulate
Ca
homeostasis in these cells. As shown in Fig. 4, NT induced a strong but transient increase in
Ca
efflux in HUVEC monolayers which had
been prelabeled with
Ca
overnight. The
maximal increase as well as the kinetics of
Ca
efflux were dose-dependent. At high concentrations of NT, the
maximal efflux was reached after 30 s of incubation, whereas at lower
concentrations it took 1 min to attain the peak response (Fig. 4). As shown in the inset of Fig. 4, the
concentration-effect relationship of NT was steep, with a threshold
around 1 nM of NT, and a maximal effect was reached at 10
nM NT. This maximal effect represented a loss of around 15% of
the total intracellular
Ca
during 1 min
of stimulation. To determine whether the functional effects of NT
analogues were related to their potency as inhibitors of
I-NT binding, these compounds were tested. As shown in Fig. 5, all NT analogues with the exception of the very low
affinity compound [D-Tyr
]NT were able
to induce
Ca
efflux from HUVECs. The
rank order of potency was identical to the order determined in binding
experiments (EC
± S.E. nM, n = 4): NT
(1.3 ± 0.23), NT (4
± 1.5), acetyl-NT
(10 ± 5),
[Phe
]NT (90 ± 20). Levocabastine had no
effect up to a concentration of 10 µM. A good correlation
was obtained between binding data and the functional response of NT
analogues (r = 0.997) with a slope value not very far
from unity (0.88). This observation strongly suggested that the
I-NT binding sites were involved in the activation of
Ca
efflux in HUVECs. To further
demonstrate the specificity of the stimulatory effect of NT, the effect
of the antagonist SR 48692 on NT-induced
Ca
release was determined. SR 48692 inhibited
Ca
efflux with an IC
value
of 8 ± 1.2 nM, a value close to its IC
value in binding experiments. NT has been shown to increase the
turnover of phosphoinositide in HT29 cells(30) , and the effect
of NT on
Ca
efflux suggests that the
peptide may also stimulate phosphoinositide turnover in endothelial
cells. Indeed, as shown in Fig. 6, NT increased phosphatidyl
turnover about 2-fold in a concentration range similar to that active
in HT29 cells and identical to the concentrations increasing
Ca
efflux in endothelial cells. Like for
Ca
efflux, this effect of NT was
inhibited in a dose-dependent manner by the antagonist SR 48692 (Fig. 6, inset).
Figure 3: Detection of the NT receptor mRNA in HUVEC. cDNA derived from HUVEC mRNA was amplified by RT-PCR, and electrophoresis was carried out as described under ``Experimental Procedures.'' Gel was stained for 6 min with ethidium bromide (1 µg/ml) and photographed under UV light (A). Hybridization was performed after transfer onto a nitrocellulose membrane and HRP-labeled probe was detected (B). Lane 1, standards; lane 2, control samples; lane 3, PCR amplification product from HUVECs.
Figure 4:
Effect of NT on Ca
efflux in HUVECs.
Ca
-labeled cell monolayers were washed
every 30 s with PSS and
Ca
in the
washing solution determined by scintillation counting. The horizontal bar denotes the presence of saline (&cjs1730;) or
NT (0.1 nM,
; 1 NM,
; 3 NM,
;
10 NM,
; 100 NM,
). RESULTS ARE THE MEAN
OF AT LEAST FOUR DETERMINATIONS ON DIFFERENT BATCHES OF CELLS. Inset, CONCENTRATION-EFFECT RELATIONSHIP OF THE EFFECT OF NT
ON
CA
EFFLUX. RESULTS REPRESENT THE
CA
EFFLUX DURING THE 1ST MIN OF
STIMULATION WITH NT. Error barsSHOW THE S.E. OF THE DATA. THE solid lineREPRESENTS A FIT OF THE SIGMOIDAL EQUATION TO THE
DATA.
Figure 5:
Effect
of NT analogues on Ca
Figure 6: Effect of NT on phosphoinositide metabolism in HUVECs. Cell monolayers were incubated for 30 min with different concentration of NT, and inositol monophosphate (IP1) accumulation was determined as described under ``Experimental Procedures.'' Results are expressed as percent increase of the control value and are the mean of four determinations performed in triplicate. Error bars represent the S.E. Inset, inhibitory effect of SR 48692, 10 nM (full bar) and 1 µM (shaded bar) on NT, 100 nM (empty bar)-stimulated inositol monophosphate accumulation. SR 48692 was present for 30 min before the addition of NT.
This work shows for the first time the existence of high
affinity functional NT binding sites in human endothelial cells of
aortic and venous origin. Up to now, NT binding sites have been
reported only in brain and gastrointestinal smooth muscle tissues of
different species. With the exception of the low affinity binding sites
of rat brain, which were inhibited by
levocabastine(28, 29) , these binding sites appeared
remarkably similar, with the same rank order of potency for different
NT analogues. Only in mast cells could a different type of NT receptor
exist: NT binding in these cells is inhibited by bradykinin at low
concentrations(20) , but because of the low affinity of NT
binding in these cells (K = 150
nM) it is not clear whether these binding sites are NT
receptors or are representative of the interaction of NT with other
membrane proteins (such as G-proteins) involved in the secretion of
histamine(2) . A similar remark applies to macrophage NT
binding sites, which also bound substance P(35) . At the
present time, three types of NT binding sites can be considered: the
high affinity, brain-type NT receptor, the low affinity brain receptor
and the low affinity mastocyte or macrophage NT binding sites. The NT
binding sites on endothelial cells described in this study resemble the
high affinity brain receptors by several different criteria: (i) a very
high affinity for NT and a rank order of potency for NT analogues
similar to the one observed in the rat brain, with NT
significantly more active than NT and
[Phe
]NT; (ii) no inhibition by levocabastine,
differentiating it from low affinity brain sites; (iii) no inhibition
by different histamine-releasing peptides such as bradykinin and
substance P, unlike mastocyte binding sites; and (iv) a single-band
hybridization of endothelial cell mRNA with a human brain NT receptor
probe. Together, these results suggest that HUVECs express a NT
receptor very similar to the high affinity type expressed in
electrically excitable cells such as neurons and smooth muscle cells.
Furthermore NT binding sites were found on aortic endothelial cells
from adult humans, suggesting that these NT receptors are not an
idiosyncrasy of fetal endothelial cells from umbilical vein, but are a
common feature of endothelial cells of human origin.
The NT binding
sites in endothelial cells were functionally coupled to intracellular
Ca release, and different NT analogues acted as
agonists with the same rank order of potency as that observed in
binding experiments. However, although the K
values determined in binding experiments and the EC
values from
Ca
efflux experiments
were closely correlated, EC
values were about 10-fold
higher than the K
values. A similar discrepancy
has already been observed in HT29 cells, where the IC
value of NT for stimulation of
Ca
efflux was 2 nM, whereas the dissociation constant of NT
in binding experiments was 0.27 nM(30) . As in HT29
cells, this was probably due to the short incubation times used in
Ca
efflux experiments compared to those
used in the binding studies.
NT stimulated phosphoinositide turnover
in HUVECs, suggesting that the effect on Ca
efflux was a consequence of the production of inositol
1,4,5-trisphosphate and subsequent stimulation of Ca
release from intracellular stores. Thus, results from binding
studies and second messenger determinations show that HUVECs express
functional NT receptors which are coupled to phosphoinositide turnover
and intracellular Ca
release. These receptors are
blocked by SR 48692 at low nanomolar concentrations, allowing this
compound to be used to probe the role of these receptors in vitro as well as in vivo. This is a relevant problem because,
up to now, the vascular effects of NT injection, in particular
hypotension and increase in membrane permeability, have essentially
been attributed to an indirect action through release of histamine from
mastocytes or interaction with innervating terminations. The detection
of functional NT binding sites in HUVECs raises the possibility of a
direct action of NT on endothelial cells of the cardiovascular system.
Possible consequences of endothelial NT receptor activation could be
(i) the release of vasorelaxating substances such as NO and (ii) the
increase of vascular endothelial permeability. Increases on
intracellular free Ca
, implicated both in the release
of NO from endothelial cells (36) and in the changes of
endothelial monolayer permeability induced by different
compounds(37) , strengthen this hypothesis.