Role of Nitric Oxide in Mediating In Vivo Vascular
Responses to Calcitonin Gene-Related Peptide in Essential
and Peripheral Circulations in the Fetus
A.S. Thakor, MA; D.A. Giussani, PhD
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Background—The role of calcitonin gene-related peptide (CGRP) in cardiovascular regulation is gaining clinical and
scientific interest. In the adult, in vivo studies have shown that CGRP-stimulated vasodilation in several vascular beds
depends, at least in part, on nitric oxide (NO). However, whether CGRP acts as a vasodilator in the fetus in vivo and
whether this effect is mediated via NO have been addressed only minimally. This study tested the hypothesis that CGRP
has potent NO-dependent vasodilator actions in essential and peripheral vascular beds in the fetus in late gestation.
Methods and Results—Under anesthesia, 5 fetal sheep at 0.8 gestation were instrumented with vascular catheters and
Transonic flow probes around an umbilical artery and a femoral artery. Five days later, fetuses received 2- and 5-␮g
doses of exogenous CGRP intra-arterially in randomized order. Doses were repeated during NO blockade with the NO
clamp. This technique permits blockade of de novo synthesis of NO while compensating for tonic production of the gas,
thereby maintaining basal cardiovascular function. CGRP resulted in potent and long-lasting NO-dependent dilation in
the umbilical and femoral circulations, hypotension, and a positive cardiac chronotropic effect. During NO blockade, the
femoral vasodilator response to CGRP was diminished. In contrast, in the umbilical vascular bed, the dilator response
was not only prevented but reversed to vasoconstriction.
Conclusions—CGRP has potent NO-dependent vasodilator actions in fetal essential and peripheral vascular beds.
CGRP-induced NO-dependent effects in the umbilical vascular bed may provide an important mechanism in the control
and maintenance of umbilical blood flow during pregnancy. (Circulation. 2005;112:2510-2516.)
Key Words: calcitonin gene-related peptide 䡲 fetus 䡲 hemodynamics 䡲 nitric oxide 䡲 vasodilation
C
alcitonin gene-related peptide (CGRP) is a 37–amino
acid peptide that was discovered in 1983 by alternative
processing of RNA transcripts from the calcitonin gene.1 It is
synthesized primarily in sensory neurones of the dorsal root
and trigeminal ganglia,2 which, in turn, extend axons centrally to the spinal cord and peripherally to various organs,
including blood vessels. In the vasculature, terminals from
these neurones innervate the smooth muscle layer,3 and
neuronal activation causes the release of CGRP, which
induces a powerful nonadrenergic, noncholinergic vasodilation. This potent dilator action of CGRP and the wide
distribution of perivascular neurones containing CGRP
throughout the cardiovascular system suggest that CGRP is in
a prime position to regulate blood flow under physiological
conditions.
In the adult, several mechanisms have been proposed for
CGRP-induced vasodilation. In vitro studies have suggested
either the activation of adenylate cyclase,4 resulting in the
opening of ATP-sensitive K⫹ channels (K⫹ATP)5 in the vascular smooth muscle, or the release of nitric oxide (NO) from
vascular endothelial cells.6,7 The finding that CGRP receptors
Clinical Perspective p 2516
are present on both the endothelium and smooth muscle cells
of the vasculature further supports the concept that the actions
of CGRP are mediated by pathways involving both endothelium-dependent and -independent mechanisms.8,9 However,
in vivo studies favor the proposal that the vasodilator actions
of CGRP are mediated, at least in part, by NO-dependent
pathways and not by activation of K⫹ATP channels.10
The increased maternal plasma levels of CGRP during
pregnancy,11 its presence in human cord and neonatal blood,
and the finding that the concentration of CGRP in fetal serum
correlates with both fetal weight and gestational age12 indicate a functional role for CGRP in prenatal life. This is further
supported by evidence from Dong and colleagues,13,14 who
have demonstrated the presence of components of the CGRP
receptor in the vascular endothelium and smooth muscle cells
of human fetoplacental vessels, and by Terenghi and colleagues,15 who have detected CGRP within the human nervous system during the first trimester of pregnancy. However,
little is known about the actions of CGRP in the fetal
vasculature. In the present study, we have tested the hypoth-
Received May 15, 2005; revision received July 6, 2005; accepted July 29, 2005.
From the Department of Physiology, University of Cambridge, Cambridge, UK.
Correspondence to Dino A. Giussani, PhD, Department of Physiology, University of Cambridge, Cambridge, CB2 3EG, UK. E-mail [email protected]
© 2005 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
DOI: 10.1161/CIRCULATIONAHA.105.562546
2510
Thakor and Giussani
esis that CGRP has important NO-dependent vasodilator
actions in fetal essential and peripheral vascular beds. The
hypothesis was tested by investigating the in vivo effects of
exogenous doses of GCRP before and during NO blockade on
changes in umbilical (UBF) and femoral (FBF) blood flow
measured by indwelling transonic flow probes in the unanesthetized late-gestation ovine fetus surgically prepared for
long-term recording.
Methods
Surgical Preparation
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All procedures were performed under the UK Animals (Scientific
Procedures) Act 1986 and were approved by the Ethical Review
Committee of the University of Cambridge. Five Welsh Mountain
sheep fetuses were surgically instrumented for long-term recording
at 120⫾2 days of gestation (term is ⬇145 days) under strict aseptic
conditions as previously described in detail.16 In brief, food, but not
water, was withheld from the pregnant ewes for 24 hours before
surgery. After induction with 20 mg/kg IV sodium thiopentone
(Intraval Sodium, Merial Animal Health Ltd), general anesthesia
(1.5% to 2.0% halothane in 50:50 O2:N2O) was maintained with
positive pressure ventilation. Midline abdominal and uterine incisions were made; the fetal hind limbs were exteriorized; and on 1
side, femoral arterial (internal diameter, 0.86 mm; outer diameter,
1.52 mm; Critchly Electrical Products) and venous (internal diameter, 0.56 mm; outer diameter, 0.96 mm) catheters were inserted. The
catheter tips were advanced carefully to the descending aorta and
inferior vena cava, respectively. Another catheter was anchored onto
the fetal hind limb for recording of the reference amniotic pressure.
In addition, a transit-time flow transducer was implanted around the
contralateral femoral artery (2R or 3S, Transonic Systems Inc) and
around one of the umbilical arteries, close to the common umbilical
artery inside the fetal abdominal cavity (4SB, Transonic Systems
Inc).17 The uterine incisions were closed in layers; the dead space of
the catheters was filled with heparinized saline (80 IU heparin per 1
mL in 0.9% NaCl); and the catheter ends were plugged with sterile
brass pins. The catheters and flow probe leads were then exteriorized
via a keyhole incision in the maternal flank and kept inside a plastic
pouch sewn onto the maternal skin.
Postoperative Care
During recovery, ewes were housed in individual pens in rooms with
a 12 hour/12 hour light/dark cycle. Here, they had free access to hay
and water and were fed concentrates twice daily (100 g sheep nuts
No. 6, H&C Beart Ltd). Antibiotics were administered daily to the
ewe (0.20 to 0.25 mg/kg IM Depocillin, Mycofarm), to the fetus
intravenously, and into the amniotic cavity (150 mg/kg Penbritinm
SmithKline Beecham Animal Health). The ewes also received 2 days
of postoperative analgesia if required (10 to 20 mg/kg oral Phenylbutazone, Equipalozone Paste, Arnolds Veterinary Products Ltd), as
assessed by their demeanor and feeding patterns. Generally, normal
feeding patterns were restored within 48 hours of postoperative
recovery. After 72 hours of recovery, ewes were transferred to
metabolic crates, where they were maintained for the remainder of
the protocol. The arterial and amniotic catheters were connected to
sterile pressure transducers (COBE, Argon Division, Maxxim Medical) and the flow probe leads to a flowmeter (T206, Transonics
Systems Inc). Calibrated mean fetal arterial blood pressure (corrected for amniotic pressure), fetal heart rate (triggered via a
tachometer from the pulsatility in either the arterial blood pressure or
blood flow signals), and mean UBF and FBF were recorded
continually at 1-second intervals with a computerized data acquisition system (Department of Physiology, Cambridge University).
While in the metabolic crates, the patency of the fetal catheters was
maintained by a slow continuous infusion of heparinized saline (80
IU heparin per 1 mL at 0.1 mL/h in 0.9% NaCl) containing antibiotic
(1 mg/mL benzyl penicillin, Crystapen, Schering-Plough, Animal
Health Division).
CGRP and Fetal Cardiovascular Regulation
2511
Experimental Protocol
After at least 5 days of postoperative recovery, all fetuses received 2and 5-␮g bolus doses of CGRP (␤-CGRP, C-1044, Sigma Chemicals) dissolved in heparinized saline via the femoral artery catheter in
a randomized order. Doses were injected over 2 to 3 seconds and
were administered after at least 15 minutes of stable baseline
recording. The CGRP doses were chosen from our own pilot
experiments and from a previous study by Takahashi and colleagues.18 In that study, treatment of late-gestation fetal sheep with a
similar dose of CGRP produced a marked vasodilation in the
pulmonary vascular bed. In the present study, exogenous doses of
CGRP were first given during a slow intravenous infusion of vehicle
(80 IU heparin per 1 mL in 0.9% NaCl) and then repeated during
blockade of NO with the NO clamp, a technique that is well
established in our laboratory.17,19 The NO clamp permits blockade of
de novo synthesis of NO while compensating for the tonic production of the gas, thereby maintaining basal cardiovascular function. In
brief, a bolus dose (100 mg/kg bolus dissolved in 2 mL heparinized
saline) of NG-nitro-L-arginine methyl ester (L-NAME; Sigma Chemicals) was injected via the femoral artery catheter. This was immediately followed by fetal intravenous infusion (5.1⫾2.0 ␮g · kg⫺1 ·
min⫺1, mean⫾SD, dissolved in heparinized saline) with the NO
donor sodium nitroprusside (SNP; Sigma Chemicals, UK) to return
fetal arterial blood pressure, heart rate, UBF, and FBF to pretreatment levels. At the end of the experimental protocol, the effectiveness of NO blockade by the clamp and the persistence of L-NAME
in the system were tested by withdrawal of the SNP infusion. A day
later, the ewes and fetuses were humanely killed with a lethal dose
of sodium pentobarbitone (200 mg/kg IV Pentoject, Animal Ltd).
The positions of the implanted catheters and flow probes were
confirmed, and the fetuses were weighed.
Blood Sampling Regimen
Before each experimental protocol, descending aortic blood samples
(0.3 mL) were taken using sterile techniques from the fetus to
determine arterial blood gas and acid base status (ABL5 Blood Gas
Analyzer, Radiometer; measurements corrected to 39.5°C). Values
for percentage saturation of hemoglobin with oxygen and blood
hemoglobin concentration were determined with a hemoximeter
(OSM2, Radiometer). In addition, blood glucose and lactate concentrations were measured by an automated analyzer (Yellow Springs
2300 Stat Plus Glucose/Lactate Analyser, YSI Ltd).
Data and Statistical Analyses
Umbilical vascular conductance (UVC) and femoral vascular conductance (FVC) were calculated by use of Ohm’s principle by
dividing mean blood flow (UBF or FBF) by mean corrected arterial
blood pressure. All measured variables were then analyzed for
normality of distribution and expressed as mean⫾SEM. For each
dose response of CGRP, baseline values for all cardiovascular
variables measured were obtained by averaging the data over the 60
seconds preceding the administration of each bolus dose. The change
in all cardiovascular variables to each dose of CGRP was first
analyzed for significance in relation to its own baseline, during either
saline infusion or NO blockade, using the significance of single
mean test. Comparisons between treatment (saline versus NO
clamp), dose (2 versus 5 ␮g), or interaction between treatment and
dose were assessed statistically with 2-way repeated-measures
ANOVA (Sigma-Stat, SPSS Inc). When a significant effect of time
or group was indicated, the post hoc Tukey test was used to isolate
the statistical differences. Differences in all cardiovascular variables
during and after removal of the NO clamp were analyzed with
Student t test for paired data. For all comparisons, statistical
significance was accepted at P⬍0.05.
Results
Fetal Arterial Blood Gas and Metabolic Status
Fetal arterial blood gas and metabolic status measured before
each experimental protocol were within normal ranges for
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October 18, 2005
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Figure 1. Cardiovascular responses to CGRP before and during
NO blockade. Bars represent the mean⫾SEM for the changes in
arterial blood pressure, heart rate, FBF, FVC, UBF, and UVC in
response to 2- and 5-␮g doses of CGRP before (▫; n⫽5) and
during (䡲; n⫽5) NO blockade with the NO clamp. Significant differences: aP⬍0.05, difference from own baseline (significance of
a single mean test); bP⬍0.05, 2 vs 5 ␮g CGRP; cP⬍0.05, before
vs during NO blockade (2-way repeated-measures ANOVA with
post hoc Tukey test).
Welsh Mountain sheep at ⬇125 days of gestation: pHa,
7.33⫾0.01; ABE, 1.0⫾1.0 mEq/L; HCO3⫺, 25.7⫾0.1 mEq/L;
PaCO2, 54.4⫾1.2 mm Hg; PaO2, 22.3⫾1.2 mm Hg; saturation,
54.8⫾3.2%; hemoglobin, 10.5⫾0.6 g/dL; lactate,
0.77⫾0.06 mmol/L; and glucose, 0.87⫾0.05 mmol/L. Neither treatment with CGRP nor fetal exposure to the NO clamp
affected basal arterial blood gas or metabolic status.
Arterial Blood Pressure and Heart Rate Responses
CGRP (2 and 5 ␮g) produced a dose-dependent decrease in fetal
arterial blood pressure (⫺7.6⫾0.8 and ⫺11.8⫾1.2 mm Hg) and
increase in fetal heart rate (66⫾8 and 106⫾5 bpm; P⬍0.05;
Figure 1). During NO blockade, hypotension (⫺1.9⫾1.2 and
⫺5.3⫾1.4 mm Hg) and tachycardia (20⫾6 and 31⫾6 bpm)
were markedly diminished by both doses of CGRP (P⬍0.05;
Figure 1), with the alteration in arterial blood pressure to 2 ␮g
CGRP showing no significant change from its own baseline
(P⬎0.05). CGRP also produced a dose-dependent effect on the
duration of the hypotensive (10.6⫾1.3 and 15.6⫾1.5 minutes)
and tachycardic (11.6⫾1.3 and 21.4⫾2.0 minutes; P⬍0.05;
Figure 2) responses, each of which was significantly shortened
during NO blockade (P⬍0.05; Figure 2).
Umbilical Hemodynamics
CGRP (2 and 5 ␮g) produced similar increases in UBF
(102⫾8 and 113⫾7 mL/min) and UVC [1.83⫾0.17 and
2.26⫾0.19 (mL · min⫺1) · mm Hg⫺1]. During NO blockade,
umbilical vasodilation was reversed to vasoconstriction in
response to both doses of exogenous CGRP [UBF, ⫺50⫾16
Figure 2. Response duration of the cardiovascular responses to
CGRP before and during NO blockade. Bars represent the
mean⫾SEM for the response duration in arterial blood pressure,
heart rate, FBF, FVC, UBF, and UVC in response to 2- and 5-␮g
doses of CGRP before (▫; n⫽5) and during (䡲; n⫽5) NO blockade with the NO clamp. Significant differences: aP⬍0.05, difference from own baseline (significance of a single mean test);
b
P⬍0.05, 2 vs 5 ␮g CGRP; cP⬍0.05, before vs during NO blockade (2-way repeated-measures ANOVA with post hoc Tukey
test).
and ⫺49⫾4 mL/min; UVC, ⫺0.78⫾0.24 and ⫺0.65⫾0.11
(mL · min⫺1) · mm Hg⫺1; P⬍0.05; Figure 1]. CGRP also
produced a dose-dependent effect on the duration of the
umbilical vasodilator response (14.2⫾2.3 and 19.9⫾1.4 minutes). This was significantly shortened after reversal to the
vasoconstrictor response during NO blockade (6.9⫾0.8 and
8.6⫾0.7 minutes; P⬍0.05; Figure 2).
Femoral Hemodynamics
CGRP (2 and 5 ␮g) produced similar increases in FBF (19⫾2
and 23⫾3 mL/min) and FVC [0.35⫾0.05 and 0.47⫾0.06
(mL · min⫺1) · mm Hg⫺1]. During NO blockade, the femoral
vasodilation to 2 ␮g of CGRP was abolished [FBF, 6⫾2
mL/min; FVC, 0.14⫾0.07 (mL · min⫺1) · mm Hg⫺1; P⬍0.05]
and markedly diminished to 5 ␮g CGRP [FBF, 11⫾1
mL/min; FVC, 0.25⫾0.04 (mL · min⫺1) · mm Hg⫺1; P⬍0.05;
Figure 1]. CGRP also produced a dose-dependent effect on
the duration of the femoral vasodilator response (12.3⫾1.3
and 17.6⫾1.5 minutes); this was significantly shortened
during NO blockade (8.0⫾1.5 and 7.9⫾1.3 minutes; P⬍0.05;
Figure 2).
An example of the individual cardiovascular responses to 5
␮g CGRP before and during NO blockade with the NO clamp
is shown in Figure 3.
Removal of the NO Clamp
Withdrawal of the SNP infusion led to a significant increase
in fetal arterial blood pressure (⌬12.8⫾1.4 mm Hg) and to
Thakor and Giussani
CGRP and Fetal Cardiovascular Regulation
2513
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Figure 3. Example of the cardiovascular responses to CGRP before and during NO blockade. An individual example of the response in
arterial blood pressure, heart rate, FBF, FVC, UBF, and UVC to 5 ␮g CGRP before and during NO blockade with the NO clamp.
decreases in fetal heart rate (⌬⫺38⫾8 bpm) and vasoconstriction in both the femoral [FVC, ⌬⫺0.32⫾0.06 (mL ·
min⫺1) · mm Hg⫺1] and umbilical [UVC, ⌬⫺1.65⫾0.27 (mL
· min⫺1) · mm Hg⫺1; P⬍0.05] vascular beds (Figure 4). This
provided evidence for the effectiveness of NO blockade by
the clamp and the persistence of the action of L-NAME
within the system until the end of the experimental protocol.
Discussion
The results of the present study show that fetal treatment with
CGRP elicits potent and long-lasting vasodilator actions in
the fetal umbilical and femoral vascular beds, hypotension,
and a positive cardiac chronotropic effect. During NO blockade, not only were these cardiovascular responses to CGRP
significantly reduced, but the umbilical dilator response was
Figure 4. Effect of NO clamp removal on
cardiovascular variables. Values represent the mean⫾SEM calculated every 30
seconds for arterial blood pressure, heart
rate, FBF, FVC, UBF, and UVC in
response to removal of the NO clamp
(arrow). Significant difference: ⴱP⬍0.05,
during vs removal of the NO clamp (Student t test for paired data).
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October 18, 2005
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reversed to vasoconstriction. These data support the hypothesis that CGRP has important NO-dependent vasodilator
actions in essential and peripheral vascular beds in the
late-gestation ovine fetus.
CGRP is one of the most potent vasodilator peptides
known,20 having a potency of ⬇10-fold greater than prostaglandins and ⬇100- to 1000-fold greater than classic vasodilators such as acetylcholine, adenosine, and 5-HT.21 Results
from the present study support the current literature and show
for the first time that in the fetus, in vivo treatment with
CGRP has potent dilator actions in the umbilical and femoral
vascular beds. Furthermore, fetal treatment with CGRP resulted in hypotension and a positive cardiac chronotropic
effect, which, unlike the vascular responses, showed significant dose-dependent effects. Hypotension is likely to be the
result of vasodilation in several vascular beds in the fetus,
leading to a reduction in total peripheral vascular resistance
and thus a fall in arterial blood pressure at a given cardiac
output. The positive cardiac chronotropic effect may be
elicited by a baroreflex response to hypotension. However, it
is also likely to involve the direct effects of the peptide on
CGRP receptors, which have been reported to be present in
both adult and fetal hearts.18,22 The CGRP receptors have
recently been cloned and are characterized as 7 transmembrane G protein– coupled calcitonin receptor-like receptors,
which are associated with 1 of 3 single transmembrane
spanning receptor activity modifying proteins (RAMP1,
RAMP2, RAMP3). The RAMPs interact via their NH2
terminus23 with the calcitonin receptor-like receptors to produce a different receptor specificity by influencing the formation of the receptor pocket by allosteric modulation.24 The
biological actions of CGRP are mediated predominantly
through the CGRP1 receptor, which is made up of the
calcitonin receptor-like receptors and RAMP1 proteins, both
of which have been demonstrated within the endothelium and
underlying smooth muscle cells of human fetoplacental
vessels.13,14
Unlike classic vasodilators, CGRP elicited cardiovascular
responses, which were particularly long-lasting. CGRP has a
half-life of ⬇7 to 10 minutes in the adult circulation,25 and its
vasodilator activity is removed after clearance by several
mechanisms, including those involving mast cell tryptase,26
neutral endopeptidases,27 matrix metalloproteinase-2,28 or
reuptake mechanisms into the sensory nerve terminal after
repolarization.29 The duration of all cardiovascular responses
showed a significant dose-dependent effect, clearly attributed
to the increased circulating concentration of CGRP at higher
doses and hence the increased time required for metabolic
disposition.
To assess the contribution of NO to the mechanisms of
action and clearance of CGRP, each dose of CGRP was
repeated during NO blockade with the NO clamp. This
technique permits blockade of de novo synthesis of NO while
compensating for the tonic production of the gas, thereby
maintaining basal cardiovascular function.17,19,30 Results from
the present study show that during NO blockade, there were
marked reductions in both the magnitude and duration of all
cardiovascular responses measured to increasing exogenous
doses of CGRP. In peripheral vascular beds in the fetus, the
dilator response to low doses of CGRP was totally abolished
during NO blockade, suggesting that at low concentrations,
the actions of CGRP are completely mediated via mechanisms involving NO-dependent pathways. In contrast, at
higher doses of CGRP, NO-independent mechanisms may
become recruited to mediate the much reduced but persistent
femoral vasodilator actions of the peptide during NO blockade. For example, CGRP has been shown to stimulate
adenylate cyclase,4 which, in turn, will increase cAMP
concentrations. This will activate protein kinase A and hence
lead to the phosphorylation and opening of K⫹ATP channels,
resulting in hyperpolarization and relaxation of the arterial
smooth muscle.5 Accordingly, an elegant study by Takahashi
and colleagues18 has reported that in another fetal “peripheral” circulation, the pulmonary vascular bed, CGRP increases
pulmonary blood flow via its specific receptor in part through
NO release and in part through K⫹ATP channel activation.
In essential fetal vascular beds such as the umbilical
circulation, several studies have shown that NO has an
important role in the mechanisms regulating blood flow
during basal and stimulated conditions.17,31 A recent study by
Dong and colleagues13 demonstrated that CGRP antagonism
in vitro diminished the dilator actions of exogenous CGRP in
the human fetoplacental vasculature. Furthermore, Thakor
and Giussani32 have shown that CGRP antagonism in vivo
diminished the umbilical hemodynamic defense response to
acute hypoxemia in the ovine fetus. The results of the present
in vivo study extend these findings to show that the potent
dilator action of CGRP in the umbilical vascular bed of the
ovine fetus is mediated exclusively by NO because, in
contrast to the femoral vascular bed, dilatation was not only
diminished but reversed to vasoconstriction during NO blockade. CGRP is known to increase noradrenergic and adrenergic sympathetic outflow after activation of various hypothalamic nuclei.33–35 Thus, it is possible that constriction of the
umbilical vascular bed after exogenous CGRP during NO
blockade may be mediated via activation of adrenoreceptors
in the umbilical vasculature;36 an effect that clearly is
overridden by the potent NO-dependent actions of the peptide
in this essential vascular bed. Alternatively, constrictor actions of CGRP during NO blockade may be similar to those
of acetylcholine in vessels once denuded of the endothelium
or in intact vessels after NO synthase blockade, as elegantly
demonstrated by Furchgott and Zawadzki37 and Librizzi and
colleagues.38 These effects of vasodilator agents after NO
blockade may be secondary to the release of other vasoactive
agents such as thromboxane, as shown for acetylcholine by
Tsuji and Cook.39
The difference in the pattern of the vascular responses to
CGRP after NO blockade between essential and peripheral
vascular beds may be attributed to whether these vessels
respond to the peptide in an endothelium-dependent or
-independent manner.40,41 Previous studies have reported that
in large conduit vessels such as the aorta, the majority of the
actions of CGRP are endothelium dependent, involving
the release of NO.6,40,42 Hence, it is no surprise that in the
umbilical artery, in essence an extension of the descending
aorta in the fetus, the actions of CGRP are largely dependent
on NO. In contrast, the femoral vasculature appears to be able
Thakor and Giussani
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to recruit other NO-independent mechanisms after NO blockade, consistent with evidence that CGRP has endotheliumindependent actions in more peripheral circulations, as reported for the isolated rat caudal artery43 and rabbit
mesenteric artery.5
The diminished cardiac positive chronotropic effect of
CGRP during NO blockade is likely due to a baroreflex of
reduced gain in response to a smaller fall in arterial blood
pressure. Alternatively, NO may also be involved in the
mechanisms by which CGRP directly affects cardiac pacemaker activity. In the heart, the influx of calcium resulting
from the transmembrane calcium current Ica has a fundamental role in pacemaker activity. Furthermore, it has recently
been shown that in isolated frog single heart cells, CGRP
dramatically increases Ica by mechanisms that cannot be
blocked by ␤-adrenergic antagonism.44 However, whether
this novel CGRP-mediated neurogenic control of cardiac
pacemaker activity involves NO-dependent pathways is
unknown.
The results of the present study also show that NO
blockade reduced the duration of all cardiovascular effects of
CGRP, implying that NO may act to inhibit the mechanism
mediating the metabolic disposition of the peptide. Although
information on the effects of NO on most mechanisms of
clearance of CGRP is unavailable, it is known that in adult
rats, NO downregulates matrix metalloproteinase-2 in aortic
vessels45 and that inhibition of endogenous NO enhances the
release of matrix metalloproteinase-2 in the heart.46
In conclusion, the data show that fetal treatment with
exogenous CGRP resulted in potent NO-dependent vasodilator actions in the umbilical and femoral vascular beds,
hypotension, and a positive cardiac chronotropic effect.
CGRP-induced dilatation in peripheral vascular beds involves
both NO-dependent and -independent pathways. In contrast,
the powerful dilator effects of CGRP in essential vascular
beds such as the umbilical circulation appear to be mediated
exclusively by NO-dependent pathways. CGRP-induced NOdependent effects in the umbilical vascular bed may provide
an important mechanism in the control and maintenance of
UBF during pregnancy.
Acknowledgments
This work was supported by the International Journal of Experimental Pathology and the Lister Institute for Preventive Medicine.
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CLINICAL PERSPECTIVE
Pregnancies complicated by chronic adverse intrauterine conditions show an enhanced umbilical vasodilator response to
acute hypoxic stress, of the type that may occur during labor and delivery, which may help to protect the compromised
fetus. The nature of this mechanism to enhance dilation in the umbilical circulation appears to involve the actions of NO
and calcitonin gene-related peptide. Here, we confirm that the umbilical circulation is highly sensitive to the NO-dependent
actions of calcitonin gene-related peptide in such a way that when NO is blocked, the dilator actions of the peptide on the
umbilical circulation are not only prevented but are reversed to constriction. Combined, past and present evidence suggests
an important role for calcitonin gene-related peptide in the maintenance of umbilical blood flow during late gestation in
normal and compromised pregnancies.
Role of Nitric Oxide in Mediating In Vivo Vascular Responses to Calcitonin Gene-Related
Peptide in Essential and Peripheral Circulations in the Fetus
A. S. Thakor and D. A. Giussani
Circulation. published online October 10, 2005;
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