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Division of Clinical and Molecular Endocrinology, Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4951; and the Department of Cancer Biology at the Cleveland Clinic Research Institute (S.C.E.), Cleveland, Ohio 44195
Address all correspondence and requests for reprints to: Dr. Marc Thibonnier, Room BRB431, Division of Clinical and Molecular Endocrinology, Department of Medicine, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, Ohio 44106-4951. E-mail: mxt10{at}po.cwru.edu
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Abstract |
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Saturation binding experiments with purified membranes of primary cultures of ECs from human umbilical vein (HUVEC), aorta (HAEC), and pulmonary artery (HPAEC) and [3H]AVP or [3H]OT revealed the existence of specific binding sites with a greater affinity for OT than AVP (Kd = 1.75 vs. 16.58 nM). Competition binding experiments in intact HUVECs (ECV304 cell line) with the AVP antagonist [125I]4-hydroxyphenacetyl-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-NH2 or the OT antagonist [125I]D(CH2)5[O-Me-Tyr-Thr-Orn-Tyr-NH2]vasotocin, and various AVP/OT analogs confirmed the existence of a single class of surface receptors of the classical OT subtype.
RT-PCR experiments with total RNA extracted from HUVEC, HAEC, and HPAEC and specific primers for the human V1 vascular, V2 renal, V3 pituitary, and OT receptors amplified the OT receptor sequence only. No new receptor subtype could be amplified when using degenerate primers. DNA sequencing of the coding region of the human EC OT receptor revealed a nucleotide sequence 100% homologous to that of the uterine OT receptor reported previously.
Stimulation of ECs by OT produced mobilization of intracellular calcium and the release of nitric oxide that was prevented by chelation of extra- and intracellular calcium. No stimulation of cAMP or PG production was noted. Finally, OT stimulation of ECs led to a calcium- and protein kinase C-dependent cellular proliferation response.
Thus, human vascular ECs express OT receptors that are structurally identical to the uterine and mammary OT receptors. These endothelial OT receptors produce a calcium-dependent vasodilatory response via stimulation of the nitric oxide pathway and have a trophic action.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Thus, the aforementioned studies suggest the presence of specific AVP/OT receptors in vascular endothelial cells whose nature, regional distribution, and intracellular signaling pathways remain to be determined. Therefore, we set out to address these issues in the present manuscript.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-6-diphenylglycoluril) was obtained from
Pierce (Rockford, IL). Fura-2/AM was purchased from
Molecular Probes, Inc. (Eugene, OR).
[125I]Na (SA, 131 mCi/ml), [3H]AVP (SA, 59
Ci/mmol), [3H]OT (SA, 32 Ci/mmol), and the
[125I]OT antagonist
D(CH2)5-O-Tyr(Me)-Thr-Tyr-Orn-vasotocin
([125I]OVTA; SA, 2200 Ci/mmol) were obtained from
New England Nuclear Life Science (Boston, MA). The peptide linear
V1 antagonist
4-hydroxy-phenylacetyl-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-NH2
(OHPhaa) was a gift from Dr. Maurice Manning, Medical College of Ohio,
and was radioiodinated ([125I]OHPhaa) with the Iodogen
technique and purified by HPLC, as previously described (8). The
nonpeptide V1 antagonist SR 49059 (batch MY10–075) was
provided by Dr. C. Serradeil-Le Gal, Sanofi Recherche (Toulouse,
France). The nonpeptide rat V1 antagonist OPC 21268 (batch
93F92M) and the V2 antagonist OPC 31260 (batch 93D96M) were
provided by Dr. J. F. Liard, Otsuka America Pharmaceutical, Inc.
(Rockville, MD).
Bis-aminophenoxyethane-N,N,N',N'-tetraacetic
acid (BAPTA), N-nitro-L-arginine
methyl ester (L-NAME), PD98059, bisindolylmaleimide I, and wortmaninn
were obtained from Calbiochem. KN-93 was purchased from
RBI (Natick, MA).
Radioligand binding assays
Purified plasma membranes of HUVECs, HAECs, and HPAECs grown in
MCDB 131 medium were obtained by homogenization and centrifugation in
Percoll gradient as previously described (9). Confluent ECs at passages
4–5 were washed twice with 10 ml ice-cold PBS and scrapped with 1 ml 5
mM Tris-HCl, 5 mM EDTA (pH 7.4), and enzyme
inhibitors (20 µg/ml phenylmethylsulfonylfluoride, 20 µg/ml
bacitracin, 10 µg/ml benzamidine, 10 µg/ml pepstatin, 10 µg/ml
antipain, 10 µg/ml leupeptin, and 10 µg/ml soybean trypsin
inhibitor). After centrifugation at 3,000 x g for 15
min at 4 C, the pellets were resuspended in buffer containing 5
mM Tris-HCl and 5 mM EDTA
(pH 7.4) with enzyme inhibitors and 250 mM
sucrose. After homogenization with 10 strokes of glass-Teflon pestle,
30% Percoll was added, and centrifugation was performed at 30,000
x g for 60 min at 4 C. The plasma membrane fraction was
centrifuged at 100,000 x g for 1 h at 4 C and
resuspended in 5 ml 50 mM HEPES, 10 mM
MgCl2, and 20% glycerol, pH 7.8. Saturation experiments
were performed with purified membranes in 250 µl PBS, 10
mM MgCl2, 0.2% BSA (pH 7.4), and increasing
concentrations of [3H]AVP with or without 1
µM unlabeled AVP or [3H]OT with or without
1 µM unlabeled OT. After incubation at 30 C for 30 min,
the membranes were filtered through Whatman GF/F filters (Clifton, NJ)
soaked in 50 mM Tris-HCl (pH 7.8) and 1% polyethyleneimine
and washed with 5 ml ice-cold 50 mM Tris-HCl (pH 7.4)
buffer. The filters were mixed with scintillation liquid, and
radioactivity was measured by liquid scintillation spectrometry
(Beckman Coulter, Inc. Palo Alto, CA; counter LS 5801;
yield, 64%). The affinity (Kd) and capacity of the
receptors were calculated using a nonlinear least square analysis
program (10). The protein concentration was measured with
Pierce’s bicinchoninic acid reagent, using ovalbumin as
an internal standard.
ECV304 cells were grown to confluence in six-well dishes and washed
twice with PBS, 10 mM MgCl2, and 0.2% BSA, pH
7.4. Competition binding experiments were performed in triplicate by
incubating the cells in the same medium with one fixed concentration of
[125I]OHPhaa or [125I]OVTA (0.30
nM) and increasing concentrations of unlabeled OT, AVP, the
V2 AVP agonists DDAVP and dVDAVP, the
nonpeptide V1 AVP antagonists SR 49059 and OPC21268, the
nonpeptide V2 AVP antagonist OPC31260, or the OT
antagonists
D(CH2)5[O-Me-Tyr2-Thr4-Orn8]vasotocin
and
D(CH2)5[O-Me-Tyr2-Thr4-Orn8Tyr9-NH2]vasotocin
for 30 min at 30 C. The cells were washed three times with ice-cold PBS
and lysed with 0.5 ml 0.2 N NaOH-1% SDS. Cell-bound
[125I]OHPhaa or [125I]OVTA radioactivity
was counted in a 
-counter. IC50 values were derived from
nonlinear least square analysis, and Ki values were
calculated using the equation of Cheng and Prusoff: Ki =
IC50/(1 + Lf/Kd).
RT-PCR experiments
The presence and type of endothelial AVP/OT receptors of several
human vascular endothelial beds were established by RT-PCR. Total RNA
was isolated from ECV304 cells as well as from HUVECs, HAECs, and
HPAECs grown up to passage 4 in 100-mm petri dishes using the RNeasy
kit from Qiagen following the manufacturer’s protocol. The RNA
aliquots (260/280 ratio, >1.8) were stored at -80 C until use. After
treatment with deoxyribonuclease I to eliminate possible DNA
contamination, the reverse transcriptase reaction was carried out by
mixing 5–10 µg total RNA previously denatured at 70 C for 10 min in
the presence of 0.5 µg/µl oligo(deoxythymidine) primer, 4 µl
5-fold concentrated RT buffer, 2 µl 100 mM
dithiothreitol, 1 µl 10 mM deoxy-NTPs, 1 µl 40 U/µl
RNasin, and 1 µl Superscript RT (200 U/µl; Life Technologies, Gaithersburg, MD). After incubation at 42 C for 50
min, then at 50 C for 10 min, the RT enzyme was inactivated by heating
at 70 C for 15 min. Thereafter, 2-µl aliquots of the RT reaction were
used for PCR reaction (initial denaturation at 95 C for 5 min followed
by 30 cycles, 95 C for 1 min, 56 C for 1 min, 72 C for 2 min, and an
additional cycle with extension at 72 C for 15 min) using specific
primers for the human V1 vascular, V2 renal,
and V3 pituitary AVP and OT receptors, as presented in
Table 1
. To eliminate possible
amplification of genomic DNA, these primers were chosen to overlap the
intronic region present between the corresponding sixth and seventh
transmembrane domains of all AVP/OT receptor sequences. Furthermore, to
look for the possibility of a new AVP/OT receptor subtype being
expressed in endothelial cells, we also conducted RT-PCR experiments
with a set of degenerate primers derived from the second and sixth
transmembrane domains, the region of highest nucleotide sequence
homology between the AVP/OT receptor subtypes. We verified with
complementary DNAs (cDNAs) coding for the various AVP/OT receptor
subtypes that all of these sets of primers amplified bands of the
appropriate sizes. Finally, as a positive control for each RNA
preparation, a 753-bp fragment of the glyceraldehyde 3-phosphate
dehydrogenase sequence was amplified (sense primer, 5'-GACCTCAACTACATGG
TCTACATG-3'; antisense primer, 5' TGTCGCTGTTGAAGTCAGAGGAGAC-3'). Double
strand nucleotide sequencing of the DNA fragments obtained by RT-PCR
was achieved by fluorescent labeling (Taq DyeDeoxy
Terminator Cycle Sequencing kit, PE Applied Biosystems,
Inc., Foster City, CA).
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Nitric oxide (NO) production assay
As NO production in vascular ECs is much less abundant than that
in macrophages, the formation of nitrites and nitrates was measured by
chemiluminescence using a Sievers (Boulder, CO) NOA280
instrument (12). Subconfluent primary cultures of HUVECs
isolated by the technique of Jaffe (13) were grown in 24-well dishes,
washed twice in Molecular Cell and Developmental Biology medium
and 2% FCS, and stimulated by 1 mM sodium nitroprusside,
100 nM bradykinin, or 1 µM OT alone or in the
presence of 4 mM EGTA and 10 µM BAPTA. After
1-min incubation at 37 C, the medium was removed and spun, and the
supernatant was kept at 4 C until chemiluminescence measurement.
cAMP production
cAMP production was measured in subconfluent monolayers of
HUVECs grown in 24-well dishes using the cAMP[125I]SPA
kit from Amersham (Arlington Heights, IL). ECs were
stimulated by 1 µM OT or 10 µM forskolin
for 15 min at 37 C in the presence of 0.5 mM
isobutyl-1-methylxanthine.
PG formation
PG production was measured in subconfluent monolayers of HUVECs
grown in six-well dishes and stimulated by 1 µM OT for 15
min at 37 C. PGE2 and 6-keto-PGF1 were
measured using enzyme immunoassay kits from Cayman Chemicals (Ann
Arbor, MI; catalog no. 514010 and 515211).
Cell proliferation assay
Cell proliferation was measured using the CellTiter 96 Aqueous
One Solution cell proliferation assay from Promega Corp.
(Madison, WI) based on the cellular conversion of the colorimetric
reagent 3,4-(5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy
phenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt into soluble formazan by
dehydrogenase enzymes found only in metabolically active, proliferating
cells. Primary cultures of HUVECs were grown to subconfluence in
96-well plates (30,000 cells/well), washed, and grown for 24 h in
200 µl MCDB 131 and 0.5% FBS. Cells were treated with 2% FBS or OT
for 24 h, followed by incubation with 20 µl dye solution for
3–5 h according to the manufacturer’s instructions. Subsequently,
absorbance was recorded at 490-nm wavelength using an enyzme-linked
immunosorbent assay plate reader (650-nm reference wavelength).
Data analysis
Nucleotide sequences were analyzed and compared using MacVector
software package (Oxford Molecular Group, Oxford, UK). Data were
expressed as the mean ± SEM. Statistical analysis was
based on Student’s t test and ANOVA. P <
0.05 was considered significant. Regression lines were calculated by
the least squares method.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). [3H]AVP and
[3H]OT bound to a single class of specific binding sites
(binding capacity, 132 ± 29 and 198 ± 36 fmol/mg protein,
respectively). As the affinity for OT is greater than that for AVP
(Kd = 1.75 ± 0.29 vs. 16.58 ± 3.15
nM, respectively), these results suggested that the
receptor expressed in human ECs belonged to an OT subtype rather than
to an AVP subtype. Similar findings were noted with purified membrane
preparations from HAECs and HPAECs (data not shown).
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Competition binding experiments with [125I]OH-Phaa and
increasing concentrations of cold AVP, the V1 vascular
antagonist SR 49050, and the V2 renal agonist
DDAVP revealed the existence of a single class of specific
receptors in ECV304 cells (Fig. 2). The
affinity constants for AVP, the V1 vascular antagonist and
the V2 renal agonist were 14, 19, and 154 nM,
respectively. These data suggested that the receptor present in ECV304
cells did not belong to the classical V1 vascular or
V2 renal subtype. Subsequently, we performed competition
binding experiments with the OT antagonist [125I]OVTA and
increasing concentrations of various AVP/OT analogs (Table 2). The affinities of OT and the two OT
antagonists for the receptor on ECV304 cells were in the nanomolar
range, whereas the affinities of AVP and its classical V2
agonists and antagonists as well as V1 antagonists were
much weaker. From these experiments, we concluded that, as in primary
cultures of vascular ECs, a single class of receptors is present on
ECV304 ECs and that this receptor belongs to the OT subtype.
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).
Nucleotide sequencing revealed that the cDNA fragment amplified with
these OT receptor primers (bp +659 to +1340) was identical to the
sequence of the human uterine OT receptor originally cloned by Kimura
et al. (14). The RT-PCR reaction with the degenerate primers
produced a band of 734 bp. The nucleotide sequence of this fragment
amplified with the degenerate primers matched only the human OT
receptor sequence, thus ruling out the presence of a new AVP/OT
receptor subtype in human ECs. These results suggest that the ECV304
ECs express an OT receptor subtype structurally similar to that present
in uterine and mammary glands.
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Subsequently, the 5'-coding region of the EC receptor was generated by
additional RT-PCR reactions with upstream primers (sense,
5'-TCAACTTTAGGTTCGCCTGC-3'; antisense, 5'-TCTTGAAGCTGATAAGGCCG-3')
derived from the human OT receptor sequence (bp -271 to +678; Fig. 4). Amplification of this GC-rich region
required the addition of 4% dimethylsulfoxide during PCR. Finally, the
whole open reading frame of the endothelial receptor was amplified by
RT-PCR using a single set of primers (sense,
5'-TCAACTTTAGGTTCGCCTGC-3'; antisense,
5'-TCATCTTCCATCATGGAGGC-3'; bp -271 to +1340). Complete nucleotide
sequencing of both strands of these DNA fragments obtained by RT-PCR
revealed a perfect match with the sequence of the human uterine OT
receptor.
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, bands of equal
size were amplified, thus suggesting that the uterine and endothelial
OT receptors share the same coding region. To rule out the possibility that the amplified OT receptor message was induced during cell culture, total RNA was obtained from primary cultures of HUVECs (first passage) and submitted to RT-PCR. Again, only the primers specific for the OT receptor led to amplification of DNA fragments of the right size (data not shown).
Signal transduction pathways of the OT receptor of human vascular
ECs
Mobilization of intracellular calcium. An increase in
[Ca2+]i is the hallmark of activation of the
vascular smooth muscle cell AVP V1 vascular receptor, the
AVP V3 pituitary receptor, and the uterine OT receptor.
Thus, we examined whether the OT receptor of human vascular ECs was
also coupled to mobilization of intracellular Ca2+. OT
stimulation of HUVECs loaded with fura-2 produced within seconds a
rapid spike followed by a more sustained mobilization of
intracellular Ca2+, with a return to the baseline within
3–5 min after the addition of OT (Fig. 5). This is similar to our previous
observations after stimulating vascular smooth muscle cells with AVP
(11). OT mobilization of intracellular calcium in HUVECs was
specifically blocked by the specific OT antagonist
D(CH2)5-O-Tyr(Me)-Thr-Tyr-Orn-vasotocin
and was reduced when extracellular calcium was chelated by EDTA (data
not shown).
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). Sodium
nitroprusside led to a dramatic increase in NO levels (48-fold
increase; P < 0.0007). As a positive control for
receptor-mediated NO stimulation, bradykinin stimulation of HUVECs
increased NO release 13-fold (P < 0.002). Stimulation
of the OT receptor of HUVECs led to NO production (17-fold rise;
P < 0.02). The effect of OT on NO production was
dramatically reduced by chelation of intra- and extracellular
calcium.
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(data not shown).
Stimulation of cell proliferation. We have shown that human
AVP and OT receptor subtypes modulate cellular proliferation (4).
Therefore, we studied the effect of OT stimulation on human vascular
endothelial cell growth. As shown in Fig. 6, OT stimulation of HUVECs resulted in a
dose-dependent proliferative response (EC50 = 0.42
nM). The effect of OT on EC proliferation was not altered
by the addition of the NO donor sodium nitroprusside, but was slightly
reduced in the presence of the NO synthase inhibitor L-NAME (Fig. 7). The protein kinase C inhibitor
bisindolylmeleimide significantly inhibited the mitogenic effect of OT,
whereas the MEK inhibitor PD98059 and the PI3 kinase inhibitor
wortmaninn had a limited effect on OT action. Chelation of
extracellular calcium by EGTA, chelation of intracellular calcium by
BAPTA, and inhibition of calmodulin kinase II by KN-93
dramatically hampered the mitogenic action of OT.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because of these shortcomings, we decided to address this issue in
human ECs by combining pharmacological and molecular biological
techniques. All radioligand binding data performed in our studies with
the natural ligands AVP and OT as well as with two AVP and OT
antagonists suggest that human vascular ECs express a single class of
high affinity binding sites that belong to the OT receptor subtype. The
same binding profile was observed in the three endothelial territories
studied, i.e. umbilical vein, aorta, and pulmonary artery.
Previous pharmacological studies performed in the rat and pig suggested
the existence of two subtypes of OT receptors, at least in the uterus
(20, 21). Two OT antagonists were found to have different effects on
the PG-releasing action of OT in rat endometrial/decidual cells,
whereas they were equally effective in blocking the uterotonic action
of OT in rat myometrial cells (20). Similarly, the OT antagonist
Dd(CH2)5[Tyr(Me)2]OVT
blocked the effect of OT in longitudinal strips of rat myometrium, but
did not inhibit the effect of OT in circular strips. We compared the
binding affinity constants of the nine AVP/OT analogs used in the
present study of human ECs and in our previous study of Chinese hamster
ovary (CHO) cells stably expressing the human uterine OT receptor (4).
A high level of correlation was found (r = 0.986; slope =
1.15; P < 0.0001), thus suggesting that the ligand
binding profile of the human EC OT receptor is similar to that of the
classical uterine OT receptor (Fig. 8).
Recent work from Fahrenholz’s laboratory indicates that alterations in
the myometrial plasma membrane cholesterol content modulates the
binding affinity of the OT receptor (22). Moreover,
progesterone was found recently to directly alter the
uterine sensitivity to OT by interfering with receptor binding and
inositol phosphate production (23). All of these observations could be
reconciled by considering the existence of a single type of OT receptor
whose ligand binding profile and signal transduction pathways are
modulated by the receptor phenotypic environment.
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In terms of signal transduction pathways, OT binding to its uterine
receptor is known to produce phospholipase C activation, calcium
mobilization, and stimulation of phosphatidyl inositol turnover (24). A
recent publication by Ohmichi et al. indicates that
stimulation of the OT receptor of human uterine myometrial cells
induces mitogen-activated protein kinase phosphorylation through a
pertussis toxin-sensitive, G protein-coupled mechanism (27). In
human myometrial cells, the OT receptor activates phospholipase Cß by
interacting with at least two types of G proteins, a member of the
pertussis toxin-sensitive Gi family and a member of the
pertussis toxin-insensitive Gq/11 family (28). The human
myometrium OT receptor also couples to the 80-kDa Gh and
can be coimmunoprecipitated with a specific anti-Gh
antibody (29).
The cellular signal transduction pathways linked to the endothelial OT receptor are presently unknown. Our data indicate that the OT receptor of human ECs is coupled to functional intracellular signals. As in the other organs where it is expressed, stimulation of the endothelial OT receptor leads to the mobilization of intracellular calcium. More specific to the vascular endothelial phenotypic environment, the OT receptor stimulates the NO-cGMP vasodilatory pathway, whereas no activation of the cAMP or PG pathways could be elicited. These findings are in agreement with the observation that in rats and dogs, the administration of L-NAME, an antagonist of NO synthase, markedly attenuated the vasodilating effect of DDAVP while not altering its effect on cAMP release (18, 30). A bradykinin antagonist or indomethacin did not affect the hemodynamic effects of DDAVP, suggesting little or no participation of bradykinin or PGs in the hemodynamic responses to DDAVP (18, 30, 31). OT stimulation of calcium mobilization seems to be instrumental in the activation of the NO pathway, as shown by the dramatic reduction of OT-induced NO release when calcium is chelated. Similarly, stimulation of the endothelial endothelinb receptor induces a transient vasorelaxation through activation of NO via a tyrosine kinase-dependent and calcium-calmodulin-dependent pathway (32).
One may wonder what is the physiological significance of the stimulation of NO production by OT in human vascular ECs. The review of the literature revealed that indeed the administration of OT produces a vasodilating effect (33). There is agreement that these events are related to a peripheral vasodilating effect, not to a direct cardiac effect of OT. The hypotensive effect of large doses of OT has been blamed for severe hypotensive episodes reported in hemorrhagic postpartum situations. In addition to its systemic hemodynamic effect, several studies have suggested that OT influences renal hemodynamics and sodium excretion at physiological concentrations. As reviewed by Conrad et al. (34), OT in the rat produces increases in GFR and effective filtration fraction as well as sodium excretion (35). Renal OT receptors are found in the glomeruli, particularly at the vascular pole and the macula densa. The signal transduction pathways of OT receptors include a rise in cytosolic calcium and activation of phospholipase C. The increased cytosolic calcium, in turn, stimulates the constitutive, calcium-calmodulin-dependent NO synthase that activates soluble guanylate cyclase to produce cGMP (36). In conscious male rats, the infusion of OT, producing a plasma concentration of 12.7 ± 3.3 pmol/liter, led to significant increases in sodium excretion, urine flow, and glomerular filtration rate (37). These alterations were specifically blocked by the coadministration of OT antagonists.
Our studies indicate that the stimulation of endothelial OT receptors leads to cellular proliferation. This finding is in agreement with our observation that the human OT receptor stably transfected in CHO cells is also mitogenic. Numerous mediators have been shown to be involved in the process of endothelial cell migration and proliferation. Obvious candidates include calcium and various kinases that are activated by the OT receptor. However, it is not known what role is played by OT-stimulated NO formation in the mitogenic effect of the endothelial OT receptor. Indeed, NO plays a key role in angiogenesis, although conflicting reports showing both angiogenic and antiangiogenic properties have been presented (38, 39, 40, 41, 42). Exogenous NO was shown to inhibit the proliferation of cultured bovine vascular endothelial cells (39), whereas endogenous and exogenous NO production could stimulate the proliferation of ECs at the microvascular level (38). In HUVECs and calf pulmonary artery ECs, NO acts as a crucial signal in the angiogenic response to basic fibroblast growth factor (42). These conflicting results can be explained by several parameters, including the use of different cell lines and cells from different vascular beds, and the experimental conditions of the mitogenic assays (various degrees and duration of serum starvation before agonist stimulation, use of various agonists, stimulation in the presence or absence of FCS, cells plated on plastic, collagen, or fibrin matrix). From our data, one may conclude that the endothelial OT receptor has trophic properties that may participate in the maintenance of the integrity of the vascular endothelial lining. The same property may be shared by other vasoactive peptides, such as angiotensin II, which potentiates vascular endothelial growth factor-induced angiogenic activity (43). The mitogenic action of OT in ECs involves several mediators, including calcium, calmodulin kinase II, and protein kinase C.
In conclusion, this is the first report about the nature and functions of a specific receptor of the AVP/OT family expressed in human vascular endothelial cells. This receptor, which is structurally identical to the uterine OT receptor, activates in a calcium-dependent fashion a vasodilatory pathway and modulates cell proliferation. Our findings, which are related to three vascular territories, do not exclude the possible existence of a second vasoconstricting AVP/OT receptor differentially expressed in other vascular beds. Based on the literature and our present findings, the intracellular pathways coupled to the endothelial OT receptor include the following. OT binding to its endothelial receptor stimulates phospholipase C to produce 1,4,5-trisphosphate, with subsequent influx and internal release of calcium. Calcium activates NOS and NO production. NO stimulates guanylate cyclase to produce cGMP, a potent activator of cGMP-dependent protein kinase G. Also, the formation of diacylglycerol stimulates protein kinase C, which, in turn, modulates the production of NO through activation of NOS. Other possibilities, such as an interaction between protein kinases C and G, may also take place and will be investigated. As a matter of fact, little is known about the interaction of G protein-coupled receptors and NO activation besides the fact that NO negatively modulates the generation of inositol 1,4,5-trisphosphate and diacylglycerol and the ensuing blunting of calcium release (44).
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Footnotes |
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Received September 14, 1998.
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 L. Vignozzi, G. B. Vannelli, A. Morelli, R. Mancina, M. Marini, P. Ferruzzi, C. Crescioli, M. Luconi, S. Donati, A. D. Fisher, et al. Identification, characterization and biological activity of oxytocin receptor in the developing human penis Mol. Hum. Reprod., February 1, 2005; 11(2): 99 - 106. [Abstract] [Full Text] [PDF]
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Y. Yamaguchi, K. Yamada, T. Suzuki, Y.-P. Wu, K. Kita, S. Takahashi, M. Ichinose, and N. Suzuki Induction of uPA release in human peripheral blood lymphocytes by [deamino-Cysl,D-Arg8]-vasopressin (dDAVP) Am J Physiol Endocrinol Metab, November 1, 2004; 287(5): E970 - E976. [Abstract] [Full Text] [PDF]
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 M. Jankowski, B. Danalache, D. Wang, P. Bhat, F. Hajjar, M. Marcinkiewicz, J. Paquin, S. M. McCann, and J. Gutkowska Oxytocin in cardiac ontogeny PNAS, August 31, 2004; 101(35): 13074 - 13079. [Abstract] [Full Text] [PDF]
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 K. Matsuura, T. Nagai, N. Nishigaki, T. Oyama, J. Nishi, H. Wada, M. Sano, H. Toko, H. Akazawa, T. Sato, et al. Adult Cardiac Sca-1-positive Cells Differentiate into Beating Cardiomyocytes J. Biol. Chem., March 19, 2004; 279(12): 11384 - 11391. [Abstract] [Full Text] [PDF]
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 J. ANTUNES-RODRIGUES, M. DE CASTRO, L. L. K. ELIAS, M. M. VALENCA, and S. M. McCANN Neuroendocrine Control of Body Fluid Metabolism Physiol Rev, January 1, 2004; 84(1): 169 - 208. [Abstract] [Full Text] [PDF]
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Y.-J. Jeng, S. L. Soloff, G. D. Anderson, and M. S. Soloff Regulation of Oxytocin Receptor Expression in Cultured Human Myometrial Cells by Fetal Bovine Serum and Lysophospholipids Endocrinology, January 1, 2003; 144(1): 61 - 68. [Abstract] [Full Text] [PDF]
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 D. Wang, J. Gutkowska, M. Marcinkiewicz, G. Rachelska, and M. Jankowski Genistein supplementation stimulates the oxytocin system in the aorta of ovariectomized rats Cardiovasc Res, January 1, 2003; 57(1): 186 - 194. [Abstract] [Full Text] [PDF]
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 M.-M. Trinh-Trang-Tan, F. Lasbennes, P . Gane, N. Roudier, P. Ripoche, J.-P. Cartron, and P. Bailly UT-B1 proteins in rat: tissue distribution and regulation by antidiuretic hormone in kidney Am J Physiol Renal Physiol, November 1, 2002; 283(5): F912 - F922. [Abstract] [Full Text] [PDF]
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G.C. Weston, I. Haviv, and P.A.W. Rogers Microarray analysis of VEGF-responsive genes in myometrial endothelial cells Mol. Hum. Reprod., September 1, 2002; 8(9): 855 - 863. [Abstract] [Full Text] [PDF]
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J. Paquin, B. A. Danalache, M. Jankowski, S. M. McCann, and J. Gutkowska Oxytocin induces differentiation of P19 embryonic stem cells to cardiomyocytes PNAS, July 9, 2002; 99(14): 9550 - 9555. [Abstract] [Full Text] [PDF]
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 P. Cassoni, A. Sapino, S. Deaglio, B. Bussolati, M. Volante, L. Munaron, A. Albini, A. Torrisi, and G. Bussolati Oxytocin Is a Growth Factor for Kaposi's Sarcoma Cells: Evidence of Endocrine-Immunological Cross-Talk Cancer Res., April 1, 2002; 62(8): 2406 - 2413. [Abstract] [Full Text] [PDF]
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M. E. Miller, S. T. Davidge, and B. F. Mitchell Oxytocin does not directly affect vascular tone in vessels from nonpregnant and pregnant rats Am J Physiol Heart Circ Physiol, April 1, 2002; 282(4): H1223 - H1228. [Abstract] [Full Text] [PDF]
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 C. L. Holmes, B. M. Patel, J. A. Russell, and K. R. Walley Physiology of Vasopressin Relevant to Management of Septic Shock Chest, September 1, 2001; 120(3): 989 - 1002. [Abstract] [Full Text] [PDF]
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 S. Mukaddam-Daher, Y.-L. Yin, J. Roy, J. Gutkowska, and R. Cardinal Negative Inotropic and Chronotropic Effects of Oxytocin Hypertension, August 1, 2001; 38(2): 292 - 296. [Abstract] [Full Text] [PDF]
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G. Bussolati and P. Cassoni Editorial: The Oxytocin/Oxytocin Receptor System--Expect the Unexpected Endocrinology, April 1, 2001; 142(4): 1377 - 1379. [Full Text]
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G. Gimpl and F. Fahrenholz The Oxytocin Receptor System: Structure, Function, and Regulation Physiol Rev, April 1, 2001; 81(2): 629 - 683. [Abstract] [Full Text] [PDF]
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P. Cassoni, A. Sapino, L. Munaron, S. Deaglio, B. Chini, A. Graziani, A. Ahmed, and G. Bussolati Activation of Functional Oxytocin Receptors Stimulates Cell Proliferation in Human Trophoblast and Choriocarcinoma Cell Lines Endocrinology, March 1, 2001; 142(3): 1130 - 1136. [Abstract] [Full Text] [PDF]
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H. Nakamura, A. Itakuara, M. Okamura, M. Ito, A. Iwase, Y. Nakanishi, M. Okada, T. Nagasaka, and S. Mizutani Oxytocin Stimulates the Translocation of Oxytocinase of Human Vascular Endothelial Cells Via Activation of Oxytocin Receptors Endocrinology, December 1, 2000; 141(12): 4481 - 4485. [Abstract] [Full Text] [PDF]
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J. F. Novak, M. B. Judkins, M. I. Chernin, P. Cassoni, G. Bussolati, J. A. Nitche, and S. K. Nishimoto A Plasmin-derived Hexapeptide from the Carboxyl End of Osteocalcin Counteracts Oxytocin-mediated Growth of Inhibition of Osteosarcoma Cells Cancer Res., July 1, 2000; 60(13): 3470 - 3476. [Abstract] [Full Text]
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M. Jankowski, D. Wang, F. Hajjar, S. Mukaddam-Daher, S. M. McCann, and J. Gutkowska Oxytocin and its receptors are synthesized in the rat vasculature PNAS, May 23, 2000; 97(11): 6207 - 6211. [Abstract] [Full Text] [PDF]
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X174 DNA HaeIII digest.
