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Division of Endocrinology and Metabolic Medicine (P.D.U., C.A., G.M.T., K.A.N., M.A.G., S.R.B., D.M.S.), Royal Postgraduate Medical School, Hammersmith Hospital, London W12 ONN, United Kingdom; and Department of Clinical Endocrinology (A.J.L.C.), St. Bartholomew’s Hospital Medical College, London, EC1A 7BE, United Kingdom
Address all correspondence and requests for reprints to: D. M. Smith, Ph.D., Division of Endocrinology and Metabolic Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 ONN, United Kingdom. E-mail: dsmith{at}rpms.ac.uk
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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CGRP binding was shown by both
ADM and CGRP (8–37), whereas CGRP and CGRP (8–37) did not compete for
125I-ADM-binding sites. The density of the ADM-binding
sites was 10 times greater in pregnant uterus (Bmax =
211 ± 39 fmol/mg protein, P < 0.01) than
nonpregnant uterus. CGRP receptor messenger RNA was identified in both
nonpregnant and pregnant uteri. In isolated nonpregnant rat uteri, CGRP
and ADM attenuated the contractile response to galanin by 77 ±
10% and 57 ± 10%, respectively. The responses to both CGRP and
ADM were abolished by CGRP (8–37). These results demonstrate, for the
first time, the presence of ADM and specific binding sites for both ADM
and CGRP in the rat uterus. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CGRP-containing primary afferent neurones predominating in the
cardiovascular system, brain and urinogenital tract but with both
CGRP and ßCGRP-containing neurons present in the gastrointestinal
tract (6, 7, 8). One of the major biological effects of both CGRP and ADM is their ability to relax smooth muscle. In the guinea pig lung, ADM acts as a potent bronchodilator (9), and CGRP has been shown to relax guinea pig trachea and rat respiratory tract smooth muscle (10, 11). ADM and CGRP are potent vasodilators in many species (12, 13, 14, 15, 16). The specific CGRP receptor subtype 1 (CGRP1) antagonist, CGRP (8–37), inhibits the vasodilator response to ADM in the isolated perfused mesenteric vascular bed (17) and in the isolated rat heart (18), indicating that the effect of ADM in these tissues is via CGRP1 receptors. However, the systemic hypotensive effect of CGRP, but not that of ADM, is blocked in vivo by CGRP (8–37) (14, 19), showing that their systemic effects are mediated by different receptors. Specific binding sites for ADM that do not bind CGRP have been identified in a number of rat tissues (20), blood vessels (14), and vascular smooth muscle cells (21). We have recently cloned a rat ADM receptor (22) that displays a similar pharmacology and mRNA distribution to the binding sites identified in rat tissues (20). In addition, we have shown that the orphan dog RDC1 receptor, when expressed in COS-7 cells, has the pharmacological characteristics of a CGRP1 receptor (23).
CGRP-IR previously has been identified in neurones innervating the
female rat urinogenital tract (7). Capsaicin treatment depletes the
majority of CGRP-IR in these tissues, indicating that this
immunoreactivity is probably CGRP localized to the primary sensory
afferent neurones (7). Immunocytochemistry of rat uterus has shown that
CGRP-IR fibres innervate the mesometrium of the uterine horn, the
mesometrial smooth muscle, and vascular smooth muscle and also are
located in the myometrium, endometrium, and endocervix of the uterine
wall (24). CGRP has been shown to inhibit spontaneous contractions in
the rat and human uterus (25, 26). Although CGRP and CGRP (8–37) do
not affect basal uterine tension in rats, CGRP elicits dose-dependent
relaxation of uterine smooth muscle precontracted with acetylcholine or
galanin (24, 27). CGRP-IR, in the neurons innervating the guinea pig
uterus, disappears during late pregnancy (28). CGRP is expressed not
only in sensory neurones but also in spinal motoneurones in the rat and
man (29, 30). In addition to its relaxant effect, CGRP derived from
motoneurones also has been proposed to act as an anterograde muscle
trophic factor. CGRP has a role in the regulation of acetylcholine
receptor gene expression at the neuromuscular junction (31) and has
been shown to increase cAMP levels in skeletal muscle (32).
Because the distributions and functions of CGRP and ADM overlap in other tissues, we hypothesized that a similar situation exists in the uterus. Before this study, the presence of ADM peptide or receptors in the uterus had not been reported. To study the possible roles of ADM and CGRP in pregnancy, we measured peptide immunoreactivity and mRNA, and receptor binding and mRNA in nonpregnant and 20-day pregnant rat uteri. Here, we show the presence of ADM-IR and mRNA in the rat uterus, as well as specific ADM-binding sites. Studies of isolated rat uterine horns demonstrate that both ADM and CGRP attenuate the galanin-stimulated contraction of smooth muscle. This indicates a possible role for ADM in the control of uterine muscle tone.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CGRP, human ADM (22–52), and human CGRP (8–37)
were synthesized using an Advanced Chemtech 396 MPS synthesizer
(Advanced Chemtech Inc., Louisville, KY). All peptides were purified by
HPLC and checked for correct molecular weight by mass spectroscopy.
Na125I and [-32P]-dCTP were supplied by
Amersham International (Little Chalfont, Buckinghamshire, UK). All
other reagents were supplied by Merck (Poole, Dorset, UK) or Sigma
(Poole, Dorset, UK). Nonpregnant female (200–250 g) and age-matched
late pregnant (20-day gestation) Wistar rats were obtained from Charles
River (Margate, Kent, UK). All procedures were performed according to
the Home Office Animals (Scientific Procedures) Act 1986.
RIA
Tissue extraction.
Whole uteri and placentae from Wistar
rats (five nonpregnant, seven pregnant) were weighed and extracted for
peptide immunoreactivity by plunging into 10 ml of 0.5 M
acetic acid per gram of tissue and boiling for 10 min. Extracts were
cooled on ice and frozen at -20 C until assay. Aliquots (100 µl) of
the extracts were dried by rotary evaporation, resuspended directly in
the relevant assay buffer, and assayed in duplicate for CGRP or rat
ADM. Values are expressed as pmol/g wet weight tissue. The peptide
contents of the nonpregnant and pregnant uteri were compared by
Student’s unpaired t test.
ADM RIA.
RIA for rat ADM was performed, using an antiserum
raised in rabbits against synthetic human ADM conjugated to BSA, using
the glutaraldehyde method (33). The antiserum (designated FC1)
cross-reacted fully with rat ADM and was used at a final dilution of
1:4,000. There was no cross-reactivity with synthetic rat CGRP,
IAPP, or calcitonin. Radioactive tracer was prepared from a synthetic
ADM (22–52) fragment (15 µg, 2.6 nmol) using the iodogen (1, 3, 4,
6-tetrachloro-3, 6-diphenylglycoluril) method, as previously
described (34). Iodinated peptide was separated by reversed-phase HPLC
(Waters C18 Novapak, Millipore, Milford, MA) using a linear
gradient of 10–40% aqueous acetonitrile containing 0.05%
trifluoroacetic acid over 80 min. The specific activity of the tracer
was 22.1 Bq/fmol. The assay was set up in a final vol of 0.7 ml [the
assay buffer comprising 0.06 M sodium phosphate (pH 7.2),
10 mM EDTA, 7 mM sodium azide, containing 0.3%
(wt/vol) BSA] and incubated at 4 C for 3 days. Bound and free tracer
were separated by dextran-coated charcoal. The detection limit was 3.5
fmol/tube at 95% confidence limits. The intra- and interassay
coefficients of variation were 8% and 12.5%, respectively.
CGRP RIA.
RIA for CGRP was as previously described (8).
The detection limit was 1 fmol/tube at 95% confidence limits. The
antiserum (CG7) (used at a final dilution of 1:112,000) cross-reacted
fully with CGRP, displayed 2% cross-reactivity with ßCGRP, and
did not cross-react with rat ADM or IAPP. The intra- and interassay
coefficients of variation were 6.6% and 10.8%, respectively.
Radioactive tracer was 125I-[Tyr0]CGRP (12
µg; 2.4 nmol) iodinated using the iodogen method, as previously
described (34). Iodinated peptide was separated by reversed-phase HPLC
using a linear gradient of 20–50% aqueous acetonitrile containing
0.05% trifluoroacetic acid over 90 min. The specific activity of the
tracer was 35.9 Bq/fmol.
Northern blot analysis
RNA preparation and Northern blotting.
Nonpregnant and
pregnant rats (n = 8) were killed and the uteri removed and pooled
in pairs (4 nonpregnant and 4 pregnant pairs). Placentae also were
collected and pooled from pairs of pregnant animals. All tissues were
frozen immediately in liquid nitrogen and stored at -80 C until
extraction. Total RNA was prepared from frozen tissues using the acid
guanidinium thiocyanate/phenol/chloroform method (35). Total RNA (50
µg) was fractionated, using denaturing MOPS/formaldehyde/1% agarose
gels, and transferred to Hybond-N (Amersham International) nylon
membranes, followed by baking at 80 C for 2 h.
cDNA probes.
Membranes were probed for rat ADM mRNA using a
150-bp cDNA probe corresponding to the entire coding sequence of rat
ADM (bases 283–432) (2). The probe was labeled using a specific
antisense primer corresponding to nucleotides 412–432 of the rat ADM
cDNA sequence. The probe for rat CGRP represents the 3' noncoding
sequence of the corresponding cDNA, as previously described (29).
Membranes were probed for rat ADM receptor mRNA using a 416-bp cDNA
probe corresponding to nucleotides 467–882 of the coding region. The
fragment used was generated by BglI digestion of a 1.2-kb
cloned fragment (22). The probe for the CGRP1 receptor was
approximately 700 bp, prepared from the rat homologue (Clark, Adrian J.
L., manuscript in preparation) of the dog RDC1 CGRP1
receptor cDNA. The sequence of the rat CGRP1 receptor probe
corresponded to bases 293-1040 of the dog CGRP1 receptor
(23). The CGRP and both receptor probes were labeled by random
hexanucleotide priming (36). All probes were labeled with
[-32P]-dCTP to a specific activity more than 7 x
108 dpm/µg.
Hybridization and quantification.
The prehybridization,
hybridization, and washing conditions were as previously described
(37). Membranes were probed for either peptide precursor mRNA or
receptor mRNA. After probing for the first mRNA, membranes were
stripped of radioactive probe by incubation in 1 x TE (10
mM Tris/HCl, pH 7.5, containing 1 mM EDTA, pH
8.0) containing 0.5% (wt/vol) SDS for 15 min at 80 C. Stripped
membranes then were probed for the second mRNA to be analyzed. Washed
membranes were exposed to Kodak XAR-5 film (IBI Ltd, Cambridge, UK) at
-70 C, then counted using a phosphorimager (PhosphorImager SF,
Molecular Dynamics Ltd., Chesham, Buckinghamshire, UK). Signals were
normalized for RNA loading by reprobing with labeled oligo(dT), as
previously described (38). Results are expressed as the ratio of the
counts obtained with the probe of interest divided by the counts
obtained with oligo(dT). The relative abundances of mRNA in nonpregnant
and pregnant uteri were compared by students unpaired t
test.
Receptor-binding assays
Membrane preparation.
Uteri from nonpregnant and pregnant
(20-day gestation) Wistar rats were frozen in liquid nitrogen.
Membranes were prepared by differential centrifugation, as previously
described (34). Briefly, tissue was homogenized in ice-cold HEPES (50
mM) buffer (pH 7.6) containing sucrose (0.25
M), soybean trypsin inhibitor (10 µg/ml), pepstatin (0.5
µg/ml), leupeptin (0.5 µg/ml), antipain (0.5 µg/ml), benzamidine
(0.1 µg/ml), aprotinin (30 µg/ml), and bacitracin (0.1 mg/ml). The
homogenates were centrifuged at 1,500 x g for 10 min
at 4 C and the supernatants centrifuged at 100,000 x g
for 1 h at 4 C. The pellets were resuspended in homogenization
buffer without sucrose and centrifuged at 100,000 x g
at 4 C for 1 h. The membranes were resuspended, as above, to a
final concentration of approximately 4 mg protein/ml and stored at -80
C.
Peptide iodination.
[Tyr0]CGRP (12 µg;
2.4 nmol) was iodinated by the iodogen method, as described in the CGRP
RIA protocol. Rat ADM also was iodinated by the iodogen method (20).
Briefly, 12.5 µg (2 nmol) of rat ADM in 10 µl of 0.2 M
phosphate buffer (pH 7.2) were reacted with 10 µg iodogen and 37 MBq
Na 125 I for 4 min at 22 C. The 125I-peptide
was purified by reversed-phase HPLC, as detailed for CGRP. Fractions
showing binding (specific activity = 10.4 Bq/fmol) were aliquoted,
freeze-dried, and stored at -80 C.
Receptor-binding assays.
For CGRP-binding assays, uterine
membranes (100 µg membrane protein) were incubated at 22 C for 45 min
with 125I-labeled [Tyr0]CGRP (1,000 Bq, 56
pM) in binding buffer (20 mM HEPES buffer, pH
7.4, containing 5 mM MgCl2, 5 mM
KCl, 1 mM EDTA, 0.25 mg/ml bacitracin, and 1
µM phosphoramidon) containing 0.1% (wt/vol) BSA, as
previously described (34). The binding site-125I-CGRP
complex was separated from free tracer by centrifugation at 15,600
x g for 2 min. Nonspecific binding was determined in the
presence of 1 µM unlabeled rat CGRP. Specific binding
is defined as total binding minus nonspecific binding. For ADM-binding
assay, uterine membranes (200 µg) were incubated for 30 min at 4 C in
CGRP-binding buffer containing 0.3% (wt/vol) BSA and 500 Bq (100
pM) 125I-rat ADM, as previously described (20).
Nonspecific binding was determined in the presence of 1
µM unlabeled rat ADM. The binding
site-125I-ADM complex was separated from free tracer, as
above.
Equilibrium competition curves were constructed in the presence of constant amounts of membrane protein and radiolabeled peptide with the concentration of the unlabeled peptide varied from 0–1 µM. Binding data were analyzed by nonlinear regression to determine the dissociation constant (KD) or absolute inhibition constant and number of binding sites (Bmax), using the Receptor-Fit program (Lundon Software, Cleveland, OH). Statistical analysis of one-site vs. two-site fits for competition curves were performed within the program by F test. Only P values less than 0.05 were considered to be significant.
Isolated rat uteri
The organ bath protocol used has previously been described (39).
Nonpregnant rat uteri were mounted in 10-ml organ baths containing
modified Krebs solution (115 mM NaCl, 1.2 mM
KH2PO4, 4.7 mM KCl, 1.2
mM MgSO4.7H2O, 25 mM
NaHCO3, 5.5 mM glucose, and 1.6 mM
CaCl2) at 30 C and pH 7.4, aerated with a mixture of oxygen
and carbon dioxide (95:5%). Recordings were made using a T3 isotonic
transducer and a Washington 400MD4C four-channel recorder (Palmer
Bioscience, Kent, UK). The preparations were equilibrated under a
resting tension of 0.5 g for a 45-min period to achieve a steady
baseline. Tissues were allowed a 20-min recovery period after each
peptide addition. The uteri were initially tested for their contractile
response by injection of 0.1 µM porcine galanin directly
into the organ bath. For all subsequent analyses, the magnitudes of
responses were measured for 3 min after addition of galanin. A second
galanin response was performed to determine whether a similar control
response was observed. After recovery, either rat CGRP (0.1
µM) or human ADM (5 µM) were applied to the
tissues, followed 1 min later by addition of galanin (0.1
µM). After washing and recovery, tissues were treated for
a third time with 0.1 µM porcine galanin to ensure that a
contractile response still occurred. This protocol was performed on six
separate uteri for each peptide. In a separate group of uteri, the
effect of addition of CGRP (8–37) before addition of CGRP or ADM
was assessed. The tissues were exposed twice to galanin, as detailed
above. After recovery, CGRP (8–37) (2 µM) was
applied, followed 1 min later by either rat CGRP (0.1
µM) or human ADM (5 µM) and followed 1 min
later by addition of galanin (0.1 µM). After washing and
recovery, tissues were treated for a third time with 0.1
µM porcine galanin to ensure that a contractile response
still occurred. This protocol was performed on six separate uteri for
each peptide. Statistical comparison of the responses to ADM and CGRP
was performed by students unpaired t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CGRP immunoreactivity
). No significant difference was detected between the
levels of ADM-IR in nonpregnant and pregnant uteri. CGRP-IR was
detected in the nonpregnant uterus at a level approximately 5-fold
greater than the ADM content. CGRP-IR could not be detected in the
pregnant uteri. No immunoreactivity for either peptide was found in rat
placental extracts.
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CGRP mRNA). The band was
approximately 1.6 kb in size, as expected (2). After correction for
differences in RNA loading by oligo(dT) hybridization, the ratio of the
signals, with nonpregnant uterus arbitrarily normalized to 1.0, are
(values are mean ± SEM): lung 0.6, nonpregnant uterus
1.0 ± 0.1, placenta 1.2 ± 0.2, and pregnant uterus 1.8
± 0.3 [pregnant uterus vs. nonpregnant (n = 4 for
each), P < 0.05]. Before oligo(dT) normalization, the
blot was stripped and reprobed for CGRP mRNA. No signal was detected
in any of these tissues after 7 days exposure, although brain stem
total RNA probed at the same time produced a strong hybridizing band
(results not shown).
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, a–c) (22) were seen in
the placenta, liver, and lung, with no bands visible in uterine total
RNA from either nonpregnant or pregnant rats (Fig. 2).
The blot was stripped and reprobed for CGRP1 receptor mRNA.
Signals were observed hybridizing to a band of the expected size of 2.2
kb in the placenta and both uterine states (Fig. 3)
(23). A band also was observed in lung but not in liver mRNA. After
correction for differences in RNA loading by oligo(dT) hybridization,
the ratio of the signals for the CGRP1 receptor mRNA, with
nonpregnant uterus arbitrarily normalized to 1.0, are (values are
mean ± SEM): nonpregnant uterus 1.0 ± 0.1, lung
1.6, pregnant uterus 1.8 ± 0.4, and placenta 2.5 ± 0.7. No
significant difference was observed between the abundance of mRNA in
nonpregnant and pregnant uteri.
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CGRP or CGRP (8–37), competed for
125 I-ADM binding in uterine membranes (Fig. 4). The KD of the binding site for ADM was
80 ± 6 pM and the Bmax was 21 ± 2
fmol/mg membrane protein (n = 3). Specific binding sites for ADM
also were characterized in membranes from pregnant uteri, with respect
to competition for 125 I-ADM by rat ADM. The KD
of the binding site for rat ADM in these tissues was 160 ± 20
pM, and the Bmax was 211 ± 39 fmol/mg
membrane protein (n = 3). Thus, the density of ADM-binding sites
was 10-fold greater in pregnant uteri, compared with nonpregnant uteri
(P < 0.01).
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CGRP, CGRP (8–37), and rat ADM competed for the
125I-CGRP-binding sites with the following order of
potency: rat CGRP more than rat ADM more than CGRP (8–37) (Fig. 5). The KD of the binding site for rat CGRP
was 140 ± 20 pM, with a Bmax of 101
± 18 fmol/mg membrane protein (n = 3). The inhibition constant
values for rat ADM and CGRP (8–37) were 1,670 ± 190 and
2,960 ± 270 pM, respectively. CGRP-binding sites also
were analyzed in membranes from pregnant uteri. The KD of
the binding site for rat CGRP in membranes from pregnant uterus was
530 ± 190 pM, and the Bmax was 153
± 5 fmol/mg membrane protein (n = 3). There was no significant
difference between the Bmax values for CGRP-binding sites
in nonpregnant and pregnant uteri.
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Isolated rat uteri
Treatment of nonpregnant uteri with 0.1 µM galanin
(n = 12) stimulated uterine contraction, as expected (27), and
this response was normalized (100%). The subsequent effects of rat
CGRP and human ADM upon galanin-stimulated contraction were
expressed as a percentage of the control response. Addition of rat
CGRP (0.1 µM) or human ADM (5 µM) to the
incubation chamber 1 min before the addition of galanin (0.1
µM) significantly reduced the magnitude of the
agonist-stimulated contraction. The reductions of the contractile
response observed were 77 ± 10% (n = 6) and 57 ± 10%
(n = 6) for rat CGRP and human ADM, respectively. A typical
tracing of the galanin (control) response and the subsequent effect of
rat CGRP/human ADM on galanin-stimulated uterine contraction is
shown in Fig. 6. A lower dose of human ADM (1
µM) had little effect upon galanin-stimulated contraction
(data not shown). After washing off the peptides and allowing the
tissue to recover, the final addition of galanin elicited contractile
responses of 60 ± 13% (after CGRP) and 74 ± 8% (after
ADM) of the magnitude of the first addition. CGRP (8–37),
administered before the addition of CGRP or ADM, resulted in
contractile responses to the subsequent galanin addition of 143 ±
20% and 126 ± 16% of the control response to galanin.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CGRP-IR in the nonpregnant uterus, similar to that previously
reported by this laboratory (7), is approximately 5-fold greater than
the ADM content. No CGRP-IR was observed in the pregnant rat uterus.
This absence of CGRP-IR is consistent with the neuronal degeneration
previously reported in guinea pigs during late pregnancy (28). Thus, it
is unlikely that CGRP has a role in the control of uterine vascular or
nonvascular smooth muscle activity in late pregnancy or at
parturition.
In contrast to the peptide content, the abundance of ADM mRNA in
nonpregnant uterus is greater (170%) than in the lung. The levels of
mRNA in the pregnant rat uterus are 1.8-fold greater than in
nonpregnant uterus (P < 0.05, students unpaired
t test). The lung contains high levels of ADM mRNA, second
only to the adrenal gland, in rat tissues. Thus, the uterus may be a
previously undetected major source of ADM. The discrepancy between the
magnitude of the differences in peptide and mRNA contents is consistent
with the concept that ADM is constitutively released by most tissues
(40, 41). Thus, in late pregnancy, in the rat, the difference between
the ADM-IR and ADM mRNA contents, when compared with those in the
nonpregnant uterus, may indicate that greater amounts of ADM are being
synthesized and released by the pregnant uterus. This correlates to a
reported increase in plasma ADM in humans during pregnancy (42). If
this is the case, up-regulation of ADM synthesis and release suggests
that ADM may play a role in the control of uterine vascular or
nonvascular smooth muscle tone in late pregnancy. The control of muscle
tone by ADM could compensate for the loss of CGRP in late pregnancy
caused by neuronal degeneration (28). No CGRP mRNA was observed in
the nonpregnant or pregnant rat uterus. In the rat, CGRP mRNA
previously has been identified in the dorsal root ganglia and spinal
motoneurones (29, 30). The peptide is synthesized in the cell bodies of
these neurones and transported to their terminals located within
tissues (43). The absence of detectable CGRP mRNA in the uterus is
consistent with this observation. Although ADM mRNA is present in the
placenta, again no mRNA was observed for CGRP. The inability to
detect immunoreactivity for either peptide in placenta could indicate
that CGRP is not produced by this tissue and ADM is produced but
released extremely rapidly.
We have identified the presence of specific and pharmacologically
distinct binding sites for ADM and CGRP in the rat uterus. The
ADM-binding sites display a high affinity for rat ADM, with little
competition by rat CGRP or CGRP (8–37). This binding profile
matches that observed in the rat ADM receptors previously characterized
in our laboratory (20). The affinity of these binding sites is similar
to the specific binding sites in the heart. The density of ADM-binding
sites was approximately 10-fold greater in the pregnant uterus,
compared with the nonpregnant uterus, indicating an extensive
up-regulation of this receptor. The detection of mRNA for the ADM
receptor in placenta, but not in the uterus, corresponds to previously
published data for poly-A+ RNA (22, 44). The presence of
specific ADM-binding sites, but no mRNA signal for the cloned ADM
receptor in the uterus in either state, indicates a possible ADM
receptor heterogeneity, as suggested by Zimmerman et al.
(45).
The CGRP-binding sites showed competition in the order of potency: rat
CGRP more than rat ADM more than CGRP (8–37). There was no
significant difference between the number of CGRP-binding sites or the
abundance of CGRP1 receptor mRNA in pregnant uterus
compared with nonpregnant tissue. There was an abundance of
CGRP1 receptor mRNA in the placenta. Recently, we have
identified the orphan dog RDC1 receptor as a CGRP1
receptor, but distribution studies of this receptor did not include the
uterus (23, 46). The presence of a CGRP receptor in human placenta with
a greater affinity for ßCGRP than CGRP has previously been
reported (47). A CGRP receptor with pharmacological characteristics of
a CGRP1 receptor recently has been identified in human
synovial tissue (48). This receptor is not present in the placenta.
However, the human receptor is more closely related to the calcitonin
receptors than the ADM receptor and shares little sequence homology
with RDC1 (49).
As a preliminary step in the identification of a role for ADM in the
uterus, we have tested the inhibitory effects of both human ADM and
CGRP upon the contractile response of uterine muscle to galanin. At
the same molar concentration of galanin and CGRP as were used for this
study, CGRP previously has been reported to inhibit the
galanin-induced contraction of uterine smooth muscle by 93% (27). We
also obtained a powerful relaxation by CGRP (77%). ADM also inhibited
galanin-induced contractions but was less effective than CGRP. We also
have shown that the effects of both CGRP and ADM are abolished by
pretreatment of rat uterine tissue with CGRP (8–37), suggesting that
the contractile effects of both peptides are mediated by
CGRP1 receptors. Hence, the role of the specific ADM
receptors identified in the uterus remains uncertain.
We have shown, for the first time, the presence of ADM peptide and
specific binding sites in the rat uterus. The data presented regarding
the presence of both ADM and CGRP peptide and receptors in the
uterus indicate that both may have a role in uterine function. In
addition, the content and mRNA of both the peptides and their receptors
differ during the late gestation phase, in comparison with uteri from
nonpregnant rats. CGRP previously has been shown to elicit
dose-dependent relaxation of uterine smooth muscle (24, 27). ADM may
have a similar relaxant role in the uterus, particularly late in
pregnancy, when ADM mRNA is increased and CGRP levels are undetectable.
A role for ADM in control of uterine vascular tone cannot be excluded,
even if the effect of ADM upon uterine smooth muscle is via
CGRP1 receptors.
However, the effect mediated by the specific ADM receptors has yet to be established.
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Acknowledgments |
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CGRP probe was obtained from Dr. S. G.
Amara. |
Footnotes |
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2 Supported by a Medical Research Council studentship.
Received November 4, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Biochem Biophys Res Commun 203:719–726[CrossRef][Medline]
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 H Watanabe, E Takahashi, M Kobayashi, M Goto, A Krust, P Chambon, and T Iguchi The estrogen-responsive adrenomedullin and receptor-modifying protein 3 gene identified by DNA microarray analysis are directly regulated by estrogen receptor J. Mol. Endocrinol., February 1, 2006; 36(1): 81 - 89. [Abstract] [Full Text] [PDF]
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C. Thota and C. Yallampalli Progesterone Upregulates Calcitonin Gene-Related Peptide and Adrenomedullin Receptor Components and Cyclic Adenosine 3'5'-Monophosphate Generation in Eker Rat Uterine Smooth Muscle Cell Line Biol Reprod, February 1, 2005; 72(2): 416 - 422. [Abstract] [Full Text] [PDF]
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C. Thota, P.R.R. Gangula, Y.L. Dong, and C. Yallampalli Changes in the Expression of Calcitonin Receptor-Like Receptor, Receptor Activity-Modifying Protein (RAMP) 1, RAMP2, and RAMP3 in Rat Uterus During Pregnancy, Labor, and by Steroid Hormone Treatments Biol Reprod, October 1, 2003; 69(4): 1432 - 1437. [Abstract] [Full Text] [PDF]
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 M. Nakatsuka, T. Habara, S. Noguchi, H. Konishi, and T. Kudo Increased Plasma Adrenomedullin in Women With Recurrent Pregnancy Loss Obstet. Gynecol., August 1, 2003; 102(2): 319 - 324. [Abstract] [Full Text] [PDF]
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I. S.S. Hwang, D. J. Autelitano, P. Y.D. Wong, G. P.H. Leung, and F. Tang Co-Expression of Adrenomedullin and Adrenomedullin Receptors in Rat Epididymis: Distinct Physiological Actions on Anion Transport Biol Reprod, June 1, 2003; 68(6): 2005 - 2012. [Abstract] [Full Text] [PDF]
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 J.B. Laoag-Fernandez, T. Maruo, P. Pakarinen, I.M. Spitz, and E. Johansson Effects of levonorgestrel-releasing intra-uterine system on the expression of vascular endothelial growth factor and adrenomedullin in the endometrium in adenomyosis Hum. Reprod., April 1, 2003; 18(4): 694 - 699. [Abstract] [Full Text] [PDF]
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 D. R. Poyner, P. M. Sexton, I. Marshall, D. M. Smith, R. Quirion, W. Born, R. Muff, J. A. Fischer, and S. M. Foord International Union of Pharmacology. XXXII. The Mammalian Calcitonin Gene-Related Peptides, Adrenomedullin, Amylin, and Calcitonin Receptors Pharmacol. Rev., June 1, 2002; 54(2): 233 - 246. [Abstract] [Full Text] [PDF]
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 V. A. Cameron, D. J. Autelitano, J. J. Evans, L. J. Ellmers, E. A. Espiner, M. G. Nicholls, and A. M. Richards Adrenomedullin expression in rat uterus is correlated with plasma estradiol Am J Physiol Endocrinol Metab, January 1, 2002; 282(1): E139 - E146. [Abstract] [Full Text] [PDF]
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 L.L. Nikitenko, N.S. Brown, D.M. Smith, I.Z. MacKenzie, R. Bicknell, and M.C.P. Rees Differential and cell-specific expression of calcitonin receptor-like receptor and receptor activity modifying proteins in the human uterus Mol. Hum. Reprod., July 1, 2001; 7(7): 655 - 664. [Abstract] [Full Text] [PDF]
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L.L. Nikitenko, I.Z. MacKenzie, M.C.P. Rees, and R. Bicknell Adrenomedullin is an autocrine regulator of endothelial growth in human endometrium Mol. Hum. Reprod., September 1, 2000; 6(9): 811 - 819. [Abstract] [Full Text] [PDF]
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 J. P. Hinson, S. Kapas, and D. M. Smith Adrenomedullin, a Multifunctional Regulatory Peptide Endocr. Rev., April 1, 2000; 21(2): 138 - 167. [Abstract] [Full Text]
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), 
),
and rat ADM (•) on 125I-rat ADM binding in nonpregnant
rat uterine membranes. Values are expressed as percentage of maximal
specific binding. Each data point is the mean of three separate assays
with assays performed in triplicate.
