VIP and PACAP display different vasodilatory effects in rabbit

coronary and cerebral arteries

Torur Dalsgaarda,*, Jens Hannibalb, Jan Fahrenkrugb, Christian Rifbjerg Larsena, Bent Ottesena

aDepartment of Obstetrics and Gynaecology 537, Hvidovre University Hospital, Kettegard Alle 30, DK-2650 Hvidovre, Copenhagen, DenmarkbDepartment of Clinical Biochemistry, Bispebjerg University Hospital, Copenhagen, Denmark

Received 7 February 2002; received in revised form 23 August 2002; accepted 26 August 2002

Abstract

Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) are closely related peptides with

wide distribution in the nervous system. The aim of the present study was to investigate functional characteristics and the influence of sex

steroids on the vasodilatory effects of these two peptides in cerebral and coronary vessels from female New Zealand White (NZW) rabbits.

The localization and concentration of VIP and PACAP in cardiovascular tissue was evaluated using immunohistochemistry and

radioimmunoassays. The vasodilatory effects of VIP and PACAP were investigated using myographs, allowing isometric tension recordings.

In order to evaluate the influence of steroid hormones, the rabbits were ovariectomized and randomized to treatment for 4 weeks with 17h-estradiol (E2), Norethindrone Acetate (NETA), E2 +NETA or placebo. Ring segments of the posterior cerebral artery, the right proximal

coronary artery and the distal left coronary artery were examined.

The highest concentrations of VIP/PACAP were observed in cerebral and coronary arteries: 5.0/5.7 and 2.8/3.5 pmol/g, respectively. The

peptides were localized in nerve fibres innervating the arteries. Both peptides produced dose-dependent vasodilatory responses in all vessels

investigated. While the effects of PACAP were identical in cerebral and coronary arterial segments, the effects of VIP displayed significant

differences (Emax, pI2, Hill-slope). Treatment with sex steroids induced no changes in the vascular effects of the two peptides.

These results indicate different mechanisms of action for the vasodilating effects of the two closely related peptides VIP and PACAP in

different areas of the coronary and cerebrovascular tree. Treatment with female sex steroids does not seem to change these mechanisms.

D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Cardiovascular diseases; Blood flow; Hormones; Peptides

1. Introduction

Vasoactive intestinal polypeptide (VIP) and pituitary

adenylate cyclase activating polypeptide (PACAP) are two

structurally closely related neuropeptides with widespread

distribution in the central and peripheral nervous system and

have a broad spectrum of biological actions [1–3]. PACAP

demonstrates 68% sequence homology with VIP in the N-

terminal 28 amino acids [4]. PACAP is present in two

amidated forms, the 38-amino-acid residues PACAP-38

and the C-terminally truncated PACAP-27, both derived

from a 175-amino-acid precursor [5].

Both VIP and PACAP-27/PACAP-38 have been shown

to be potent vasodilators in several species. PACAP acts via

three types of receptors, of which one is specific for PACAP

(PAC1) and two are shared with VIP (VPAC1 and VPAC2)

[6]. In humans, VIP dilates cerebral [7,8] and coronary

arteries [9,10]. Correspondingly, VIP has been shown to

dilate rabbit cerebral arteries [11] and rabbit coronary

arteries [12]. VIP-immunoreactive neuronal cell bodies have

been located to the interatrial septa of the heart [12]. Both

PACAP-27 and PACAP-38 dilate human coronary arteries

[13,14], but PACAP-27 has been found not to affect human

middle cerebral arteries [15]. However, our knowledge of

the effect of VIP and PACAP on the arteries in the heart and

brain is still limited.

Previously, it has been observed in animals that treatment

with estradiol increases the expression of all pre-pro-VIP-

derived peptides except PHI/PHV [16], and in postmeno-

pausal women, it has been shown that the vasodilatory effect

of VIP on vaginal blood vessels is dependent on the

presence of oestrogen [17]. Furthermore, the vasomotor

0167-0115/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

PII: S0167 -0115 (02 )00205 -7

* Corresponding author. Tel.: +45-36-32-33-76; fax: +45-36-32-37-23.

E-mail address: torur.dalsgaard@dadlnet.dk (T. Dalsgaard).

www.elsevier.com/locate/regpep

Regulatory Peptides 110 (2003) 179–188

responses of isolated rabbit coronary arteries to VIP have

been demonstrated to change with age and the development

of sexual maturity, indicating a relationship with sex ste-

roids [18].

Due to the presence and significant cardiovascular effects

of VIP in the heart, the peptide has recently been suggested

to be important in the regulation of coronary blood flow

[19]. The mechanism of the putative beneficial cardiovas-

cular effects produced by estradiol in postmenopausal

women might therefore involve VIP and/or PACAP.

In non-hysterectomized women, postmenopausal hor-

mone replacement therapy (HRT) consists of a combination

of oestrogen (as 17h-estradiol) and progestogen (as the

synthetic compound Norethindrone Acetate). These are

female sex steroids, inducing their action mainly by chan-

ging the transcription of DNA in the cell nucleus via

specific intracellular steroid receptors. Phytoestrogens are

plant-derived molecules possessing oestrogen-like activity,

most of them with the ability of inducing different estro-

genic effect in different cells and therefore also termed

selective estrogen receptor modulators (SERMs).

Based on this, the aim of the present study was to

determine the presence of VIP and PACAP in cardiovascu-

lar tissue and to examine the vascular effects of the two

peptides on cerebral and coronary arteries from normal and

sex steroid-treated rabbits [20].

2. Materials and methods

2.1. Animals

Material and methods are described earlier in detail for a

similar study of vascular function in atherosclerotic rabbits

[21]. In short, the rabbits used in the present study were

New Zealand White (NZW) rabbits (SSI:CPH), which

during the study were housed individually in stainless steel

cages under regulated environmental conditions. Protocols

met the Danish Regulations for Animal Research and all

experiments were carried out as investigator blinded studies.

2.2. Tissue extraction and radioimmunoassays (RIA)

Six female rabbits at the age of 22 weeks were anae-

sthetized (pentobarbital 100 mg/kg intravenously). The

abdomen and thorax were cut open and the entire heart

with aorta, the iliac arteries and the inferior caval vein were

removed. The head was separated from the body, the skull

was carefully opened with an oscillating saw and the entire

brain with the brainstem was removed. The tissue was

rapidly placed in ice-cold Krebs buffer (NaCl 119 mM,

KCl 4.6 mM, NaHCO3 15 mM, CaCl2 1.5 mM, MgCl2 1.2

mM, NaH2PO4 1.2 mM and glucose 11 mM) to reduce

metabolism and anoxia. All Krebs buffer solutions were

constantly bubbled with 5% CO2 + 95% O2 to maintain pH

at 7.4. Throughout the subsequent dissection, the tissue was

bathed in cold Krebs buffer at 4 jC. The cerebral arteries ofthe posterior part of the circle of Willis (the posterior

communicating arteries, the posterior cerebral arteries and

approximately 15 mm of the basilar artery) were isolated

from the ventral surface of the brain by careful micro-

dissection under a stereomicroscope and dissected free of

connective tissue. The intramural part of the left anterior

descending coronary artery (f 20 mm) was isolated and

freed of fat tissue, ventricular muscle and connective tissue.

A square of the myocardium (f 5� 5 mm, without visible

coronary arteries) was cut from the left ventricle near apex.

Furthermore, ring segments (f 20 mm in length) were cut

from the thoracic aorta, the caval vein and the proximal iliac

arteries (bilaterally, f 10 mm from each side). All samples

were immediately stored at � 80 jC. Before peptide ana-

lysis, the frozen tissue specimens were weighed and extrac-

ted in boiling water/acetic acid [22]. The extracted samples

were reconstituted and analyzed by radioimmunoassays

specific for PACAP-38 (antiserum code no. 733C) [22]

and VIP (antiserum code no. 5603) [23,24], respectively.

The radioimmunoassays were specific for the respective

peptides and did not show any cross-reactivity with structur-

ally related peptides. The within- and between-assay coef-

ficients of variation were below 10%. The concentrations

were expressed as pmol neuropeptide per gram wet weight

tissue (n = 6).

2.3. Immunohistochemistry

Four adult male rabbits were used for the immunhisto-

chemical analysis. On the day of tissue preparation, the

rabbits were killed by an overdose of pentobarbital intra-

cardially (500 mg/animal), which does not seem to affect

cardiovascular tissue. The brain and heart were removed,

and the major extracerebral arteries on the ventral surface of

the brain were identified using a stereomicroscope and

carefully dissected from the brain. The vessels and the heart

were placed in ice-cold Stefanini fixative for 16–24 h at 4

jC. The heart tissue was cryoprotected for 24–48 h in 30%

sucrose at 4 jC, then frozen at � 20 jC before it was cut in

12-Am-thick sections on a cryostat. The blood vessels were

placed in cryoprotectant at � 20 jC and reacted as whole

mount as described below. Double immunohistochemistry

was performed as previously described [25] using a well-

characterized mouse monoclonal PACAP antibody (code

no. JHH1, supernatant diluted 1:5) [22] and a guinea pig

anti-VIP antiserum (code no. B-GP340-1, obtained from

Euro-Diagnostica, Malmo, Sweden, diluted 1:500). PACAP

and VIP immunoreactivity were visualized using a biotiny-

lated donkey anti-mouse antiserum (code no. 115-065-151,

diluted 1:800, Jackson Immunoresearch Laboratories, West

Grove, PA, USA) and FITC-conjugated donkey anti-guinea

pig antiserum (code no. 706-045-148, diluted 1:50) in

combination with biotinylated tyramide (Tyramide System

amplification, DuPont NENR, Boston, MA, USA) and

streptavidin–Texas RedR. As controls, sections and/or

T. Dalsgaard et al. / Regulatory Peptides 110 (2003) 179–188180

whole mounts were routinely incubated with antibodies

preabsorbed with the respective antigen (20 Ag/ml), which

abolished all staining. Images were grabbed via a Leica

DC200 camera using Leica DC200 software (Leica, Cam-

bridge, UK). Image editing software (Adobe Photoshop/

Adobe Illustrator) was used to combine the obtained images

into plates, and figures were printed on a Tektronix (Wilson-

ville, OR) Phaser 450 dye sublimation printer.

2.4. Functional studies

2.4.1. Surgery and experimental design

Following 1 week of acclimatisation, at the age of 16

weeks, 32 female rabbits were bilaterally ovariectomised

during anaesthesia with 0.4 ml/kg body weight of a mixture

of Hypnorm (fentanyl citrate 0.315 mg/ml + fluanisone 10

mg/ml, Janssen-Cilag, Sauderton, High Wycombe, Buck-

inghamshire HP14 4HJ, UK), Dormicum (5 mg/ml, F.

Hoffman-La Roche, Basel, Switzerland) and sterile water

(1:1:2). After 2 weeks of recovery, from the age of 18

weeks, the rabbits were randomized to four different groups

of treatment (each group n = 8): placebo control, 17h-estra-diol (E2), Norethindrone Acetate (NETA) or E2 +NETA. To

avoid the influence of phytoestrogens, the rabbits were fed

100 g/animal/day of semisynthetic pelleted diet C2000

(Altromin International, D-4937 Lage, Germany) during

the recovery and treatment periods. Furthermore, the batch

of semisynthetic chow was tested for the content of the

major isoflavonoids commonly found in soy, using extrac-

tion with water/methanol/formic acid followed by analysis

with high-pressure liquid chromatography (HPLC) and

liquid chromatography/mass spectrometry (LCMS). No

traces of these phytoestrogens were found in the semi-

synthetic chow. During the 4-week treatment period, the

concentrations of E2 and NETA (both Novo Nordisk Far-

maka, Lyngby, Denmark) added to the chow were 0% and

0% for the placebo group, 0.004% and 0% for the E2 group,

0% and 0.003% for the NETA group and 0.004% and

0.003% for the E2 +NETA group, respectively. One rabbit

from the E2 +NETA group suffered from diarrhoea for the

last 2 weeks of the treatment period and displayed oedema

of the gut and the brain at dissection. Consequently, this

rabbit was excluded from the study.

2.4.2. Tissue preparation and myograph experiments

At the age of 22 weeks, two rabbits from different groups

of treatment were anaesthetized (pentobarbital 100 mg/kg

intravenously). The entire heart and brain were removed,

and the cerebral and coronary arteries were microdissected

as described above. Ring preparations (f 1 mm in length)

of three artery segments were prepared for the myograph

experiments: from the middle part of the posterior cerebral

artery (PCA), from the proximal epicardial part of the right

coronary artery as close to its origination from aorta as

possible (PROX) and from the intramural part of the left

anterior descending coronary artery (LAD), halfway

between aorta and apex of the heart. One LAD segment

from the placebo group and two PROX segments (from the

placebo and E2 group, respectively) could not be dissected.

Blood samples (nz 7 for all groups) were analyzed for E2

and estrone [26]. Two 40-Am wires were carefully inserted

through the lumen of each artery ring preparation for

mounting in double myographs [27] (Myo-Interface 500A,

JP Trading, Aarhus, Denmark), allowing measurement of

the isometric tension relations. All the experiments were

performed with two corresponding artery segments from

two differently treated rabbits, which were assigned ran-

domly and mounted on the near or far transducer in the

same double myograph, thus allowing the two arteries to be

tested and compared simultaneously and exposed to the

same solutions. The relation between resting wall tension

and internal circumference was determined, and from this,

the internal circumference, L100, corresponding to a trans-

mural pressure of 13.3 kPa ( = 100 mm Hg) for a relaxed

vessel in situ was calculated [28]. The vessels were set to the

internal circumference Ll = 0.9� L100. Previously, it has

Table 1

Characteristics of the rabbits used in this investigation

Treatment Placebo E2 NETA E2 +NETA ANOVA

Number 8 8 8 7a –

Body weight, start of treatment (kg) 3.3 (0.1) 3.3 (0.1) 3.2 (0.2) 3.3 (0.1) NS

Body weight, end of treatment (kg) 3.7 (0.1) 3.7 (0.1) 3.4 (0.2) 3.6 (0.1) NS

Avg. weight gain during treatment (g/week) 102 (6) 98 (15) 71 (13) 88 (16) NS

Avg. intake, E2 [mg (kg bw)� 1 day� 2] 0 (0) 1.1 (0)*,** 0 (0) 1.0 (0.1)*,** p< 0.001

Avg. intake, NETA [mg (kg bw)� 1 day� 2] 0 (0) 0 (0) 0.8 (0)*,*** 0.8 (0)*,*** p< 0.001

Serum–E2 at endpoint (pmol l� 1) 39 (0) 101 (10)*,** 39 (0) 106 (8)*,** p< 0.001

Serum–Estrone at endpoint (pmol l� 1) 53 (3) 123 (25)b 84 (11)c 122 (10)* p< 0.01

Values are mean (S.E.M.). E2 = 17h-estradiol, NETA=Norethindrone Acetate. Avg. = average, bw= body weight.a One rabbit from the E2 +NETA group was excluded because of diarrhoea and pathology at dissection.b Indicates borderline significance ( p= 0.14) compared to placebo (Student’s t-test [Bonferroni correction]).c Indicates borderline significance ( p= 0.10) compared to placebo (Student’s t-test [Bonferroni correction]).

* Indicates p< 0.001 compared to placebo.

** Indicates p< 0.001 compared to NETA.

***Indicates p< 0.001 compared to E2.

T. Dalsgaard et al. / Regulatory Peptides 110 (2003) 179–188 181

been shown that at Ll, the force production in vascular

smooth muscle is close to maximum, also in rabbit coronary

and cerebral arteries [29]. By changing the normal Krebs

buffer with 124 mM K+–Krebs buffer, which is normal

Krebs buffer with KCl exchanged for NaCl on an equimolar

basis, repeated contractions were induced until reproducible

responses were obtained, accepting a variation of V 10%. In

vessels precontracted with 30 mM K+–Krebs buffer (com-

position as normal Krebs buffer, except KCl, 30 mM, and

NaCl, 93.7 mM) and allowed to equilibrate for 20 min to

reach a plateau, the cumulative dose–response curves to

VIP and PACAP-27 (Peninsula Laboratories, St. Helens,

UK, both 10� 10–10� 6 M) were established (half-logarith-

mic steps, 3 min between each addition). With an estimated

intracellular [K+] of 150 mM, the change in extracellular

[K+] from 4.6 to 30 mM would change the membrane

potential (Vm) from approximately � 93 to approximately

� 43 mV, calculated using the Nernst equation, Vm =

RT(zF)� 1ln(Co/Ci), where R is the gas constant ( = 8.314 J

K� 1 mol� 1), T the absolute temperature (310 K= 37 jC in

the myograph bath), z the number of charges for the ion

(= + 1), F the Faraday constant ( = 9.65 104 A s mol� 1), Co

and Ci the molal [K+] outside and inside the cell membrane,

respectively. In pilot studies, we observed that the vaso-

dilating effect of PACAP-27 was more pronounced than for

PACAP-38. Earlier studies have indicated that concentration

and effect of neuropeptides are independent parameters; for

instance, it has been demonstrated that sex steroids can

influence the motor effect of VIP in smooth muscle without

changing the VIP concentration [30]. Therefore, it was

decided to use PACAP-27 for the functional studies, but

PACAP-38 for the RIA studies, since the tissue concen-

trations of PACAP-38 are considerably higher compared to

PACAP-27. Artery segments with a maximum force deve-

lopment of less than 1 mN/mm following depolarization

with 124 or 30 mM potassium were discarded. Following all

the mentioned criterions of exclusion, nz 6 for all groups

and for all types of vessels in all experiments.

2.5. Data, calculations and statistical analysis

Responses of the vessels were measured as force (F)

and expressed as active wall tension (T), which is F

divided by twice the segment length [28]. Internal lumen

diameter was calculated as l = L/p. The effective active

pressure (yPK+, max) corresponding to the maximum active

tension response to 124 mM potassium (TK+, max) was

calculated as yPK+, max = TK+, max/(Ll/2p). By computerized

iteration (GraphPad Prism Software, version 2.00, San

Diego, CA), the responses to the cumulative addition of

the peptides were fitted to sigmoidal dose–response curves

with variable Hill-slopes, used to determine the maximum

relaxant effect (Emax) and the concentration required to

produce half the maximum response (EC50), the latter

expressed as the negative logarithm to the molar concen-

tration, pI2 =� log(EC50). Results are expressed as mean

F S.E.M. Significance of difference between the treatment

groups was assessed by one-way ANOVA. If this test

indicated statistical significance, Student’s two-tailed t-test

was used to compare groups two by two, using the

Bonferroni correction for multiple comparisons. Statistical

significance was set at p < 0.05.

3. Results

3.1. Characteristics of the rabbits

Baseline values, body weight at arrival, before and after

the treatment period, average weight gain per week during

treatment and relative feed intake during the study demon-

strated comparability of the four groups of treatment (Table

1). The rabbits receiving active treatment with E2 and

E2 +NETA displayed E2 levels three times that of the placebo

and NETA group (Table 1). An average relative intake of 1.1

mg E2/kg body weight/day for the E2 group and of 1.0 mg E2/

kg body weight/day for the E2 +NETA group resulted in

physiological levels of E2 in the blood of these two groups.

The placebo and NETA group had no intake of E2, and

serum–E2 levels at euthanization for these two groups were

at the detection limit of 40 pM. Consequently, serum–E2

levels for both the E2 and E2 +NETA group were signifi-

cantly higher ( p < 0.001, Student’s t-test with Bonferroni

Fig. 2. Fluorescence photomicrographs of PACAP (A and C) and VIP immunoreactivity (B) in whole mount preparation of cerebral arteries isolated from the

basal surface of the rabbit brain (A and B) and in coronary arteries (C). Note that VIP is located in a subpopulation of PACAP containing nerve fibres

(exemplified by arrows in A and B). PACAP immunoreactive nerve fibres were found in coronary arteries located in the adventitia and media (arrows in C). V

indicates lumen of a coronary artery. Scale bars: (A) and (B) = 100 Am; (C) = 50 Am.

Fig. 1. Concentration of immunoreactive VIP (single hatched columns) and

PACAP-38 (double hatched columns) in rabbit cerebral arteries (CER), left

anterior descending coronary arteries (COR), myocardium (MYO), thoracic

aorta (AO), iliac arteries (IA) and caval vein (CV). Values (pmol/g wet

weight) are given as mean, horizontal bars indicate S.E.M. (n= 6).

T. Dalsgaard et al. / Regulatory Peptides 110 (2003) 179–188182

T. Dalsgaard et al. / Regulatory Peptides 110 (2003) 179–188 183

correction) compared to both the placebo and NETA group.

The average relative intake of NETA was 0.8 mg/kg body-

weight/day for both the E2 +NETA and NETA group. The

placebo and E2 group had no intake of NETA. No laboratory

kit was available for measuring the concentration of NETA,

but serum–estrone can be regarded as an indicator of the

NETA uptake. NETA and other progestogens induce an

increased activity of P450-aromatase [31] and 17h-hydrox-ysteroid dehydrogenase [32,33] in extra-ovarian tissue,

thereby increasing the conversion to estrone. Accordingly,

serum–estrone was significantly higher in the E2 +NETA

group compared to the placebo group ( p < 0.001, Student’s t-

test with Bonferroni correction). Estrone levels for the NETA

and E2 group were also higher compared to the placebo

group, thus confirming the intake of NETA from the chow

in the NETA group, but with a larger variation. Therefore,

the p-values were only borderline significant following

Bonferroni correction of the results from Student’s t-test

( p = 0.10 and 0.14, respectively).

3.2. Radioimmunoassays

The concentration of immunoreactive VIP and PACAP-

38 was significantly higher in coronary and cerebral

arteries compared to both myocardium, thoracic aorta, iliac

arteries and caval vein, where the concentrations were

negligible. Although the concentrations of VIP tended to

be lower than the concentrations of PACAP in the coronary

and cerebral arteries, they were not significantly different

(Fig. 1).

3.3. Immunohistochemistry

3.3.1. Basal arteries of the brain

Whole mount preparation of the basal arteries showed a

distinct pattern of PACAP immunostaining consisting of a

well-developed plexus of perivascular nerve fibres observed

Table 2

Functional characteristics for the vessels used for myograph investigations

in this study

Treatment Placebo E2 NETA E2 +NETA

PCA, ll (Am) 444 (9) 475 (30) 439 (21) 488 (26)

PCA, yTK+, max

(mN mm� 1)

2.5 (0.3) 3.2 (0.4) 2.8 (0.3) 2.8 (0.4)

PCA, yPK+, max

(mm Hg)

85 (10) 98 (12) 95 (11) 86 (10)

PCA, first

K(30/124)+ ratio (%)

113 (9) 95 (3) 92 (5) 104 (11)

PCA, second

K(30/124)+ ratio (%)

91 (11) 81 (6) 86 (7) 95 (7)

LAD, ll (Am) 790 (61) 744 (63) 596 (87) 848 (49)

LAD, yTK+, max

(mN mm� 1)

6.2 (1.2) 5.5 (0.7) 3.7 (0.9) 5.3 (0.6)

LAD, yPK+, max

(mm Hg)

121 (23) 114 (14) 87 (12) 96 (11)

LAD, first

K(30/124)+ ratio (%)

97 (6) 97 (9) 109 (9) 93 (9)

LAD, second

K(30/124)+ ratio (%)

100 (9) 101 (8) 121 (9) 96 (14)

PROX, ll (Am) 884 (64) 890 (78) 899 (102) 916 (33)

PROX, yTK+, max

(mN mm� 1)

4.1 (0.7) 5.3 (0.6) 4.2 (0.3) 4.2 (0.7)

PROX, yPK+, max

(mm Hg)

69 (9) 94 (6) 76 (8) 69 (11)

PROX, first

K(30/124)+ ratio (%)

94 (10) 96 (10) 93 (10) 107 (5)

PROX, second

K(30/124)+ ratio (%)

99 (9) 96 (12) 100 (12) 112 (7)

Values are mean (S.E.M.). PCA=posterior cerebral artery, LAD= left

anterior descending coronary artery, PROX= proximal right coronary

artery. ll = normalized effective lumen diameter; yTK+, max =maximum

active wall tension at ll for 124 mM K+ response; yPK+, max =maximum

effective active pressure at ll for 124 mM K+ response; K+(30/124)

ratio=(maximum active wall tension for 30 mM K+ response)/(yTK+, max)

ratio in percent, first ratio before the VIP experiment, second ratio before

the PACAP-27 experiment. ANOVA, all NS.

Fig. 3. Maximum vasodilatory effect (Emax, percentage of precontraction

induced by 30 mM potassium–Krebs buffer, top panel) and pI2[� log(EC50), lower panel] for VIP (panel to the left) and PACAP-27

(panel to the right) in rabbit posterior cerebral artery (PCA), left anterior

descending coronary artery (LAD) and proximal right coronary artery

(PROX). White columns = placebo group, single hatched columns = 17h-estradiol (E2) group, double hatched columns =Norethindrone Acetate

(NETA) group and black columns =E2 +NETA group. Values are mean

with S.E.M. shown as vertical bars. ANOVA, all NS (nz 6).

T. Dalsgaard et al. / Regulatory Peptides 110 (2003) 179–188184

in the wall of all major cerebral arteries and their branches

(Fig. 2A). VIP immunostaining was less intense compared

to the PACAP staining (Fig. 2B) and seemed to be located in

a subpopulation of the PACAP-immunoreactive nerve fibres

(Fig. 2A and B).

3.3.2. Coronary arteries

Few densely stained PACAP-immunoreactive nerve

fibres were found in coronary arteries located in the adven-

titia and media (Fig. 2C). VIP could not be demonstrated in

rabbit coronary arteries using immunohistochemistry with

the VIP guinea pig antiserum.

3.4. Functional studies

3.4.1. Functional characteristics of the vessels

There were no differences between the treatment groups,

for any of the vessels, in the number of steps at the

normalization procedure. The functional characteristics of

the vessels used in this investigation are summarized in

Table 2. For all vessels, activation by 124 mM potassium

induced an initial fast increase in tension, most pronounced

in the coronary arteries, which declined within 10 s to a

tonic contraction. The normalized effective lumen diameter

(ll) and the amplitude of the maximum potassium-induced

contraction (yTK+, max and yPK+, max) at ll displayed no

differences between the four groups of treatment. In all

vessels, activation by 30 mM potassium induced a slowly

developing contraction, which reached a plateau with a tonic

contraction in approximately 10–15 min. No differences

were observed between the treatment groups for any of the

vessels for the first or second 30 mM potassium response

(before the VIP and PACAP experiment, respectively) and

neither for the first and second K30/120+ ratio (Table 2).

Fig. 4. Concentration– response curves for relaxation induced by VIP

(graph A) and PACAP-27 (graph B), both 10� 10–10� 6 M (half-

logarithmic steps), in rabbit posterior cerebral arteries (closed, black

circles), distal left coronary arteries (closed, black triangles) and proximal

right coronary arteries (open circles) for pooled data (26V nV 30). Data are

expressed as percentage of the precontraction induced by Krebs buffer with

potassium, 30 mM, at the beginning of each experiment. Symbols indicate

mean with S.E.M. shown by vertical bars. *** indicates p< 0.0001 for Emax

(ANOVA).

Fig. 5. Maximum vasodilatory effect (Emax, percentage of precontraction

induced by 30 mM potassium–Krebs buffer, top panel), pI2 [� log(EC50),

middle panel] and Hill-slopes (lower panel) for VIP (panel to the left) and

PACAP-27 (panel to the right) in rabbit posterior cerebral artery (PCA), left

anterior descending coronary artery (LAD) and proximal right coronary

artery (PROX) for pooled data (26V nV 30). Values are mean with S.E.M.

shown as vertical bars. *** indicates p< 0.001, ** indicates p< 0.01 and *

indicates p< 0.05 (Student’s t-test [Bonferroni correction]).

T. Dalsgaard et al. / Regulatory Peptides 110 (2003) 179–188 185

3.4.2. Myograph dose–response experiments, VIP and

PACAP-27

Both VIP and PACAP-27 induced a dose-dependent

vasodilatory response in cerebral as well as coronary arteries.

The computerised iterations showed that treatment with sex

steroids induced no changes in Emax, pI2 or Hill-slopes in any

of the vessels for any of the two examined peptides (Fig. 3).

In general, there was a similar pattern for each treatment

group: that the VIP response was different for each vessel

type, with the smallest response in PROX, larger in LAD and

largest in PCA, while for PACAP-27, the response was

practically similar in all three artery segments. To further

examine these variations, and since treatment induced no

changes, the results for all four treatment groups were pooled

(Fig. 4). The differences between the vessel segments in

Emax, pI2 and Hill-slopes for the VIP response were all

statistically significant (ANOVA, p < 0.0001, p < 0.0001

and p < 0.05, respectively, Fig. 5). For the PACAP response,

on the contrary, Emax, pI2 and Hill-slopes were similar in all

three examined artery segments.

4. Discussion

The present study demonstrates that rabbit cerebral

arteries are supplied by VIP- and PACAP-containing nerve

fibres, and in contrast to previous findings in rat [34], the

two peptides were partly co-localized and VIP immuno-

staining was less intense compared to the PACAP staining.

Only PACAP-immunoreactive fibres were demonstrated in

the coronary arteries. Due to the species, we had to use a

guinea pig antiserum rather than our highly sensitive rabbit

anti-VIP antiserum, which could explain the inability to

detect VIP in the coronary arteries at the immunohistoche-

mical level. Both VIP and PACAP were able to produce a

vasodilatory response, but while the vasodilatory effect of

VIP differed from one vascular bed to another, PACAP

displayed identical vascular responses in all vessel seg-

ments. This observation is in accordance with a previous

study in atherosclerotic cerebral and coronary arteries from

Watanabe Heritable Hyperlipidemic (WHHL) rabbits (Dals-

gaard et al., unpublished data). The finding is unexpected,

since only three receptors for VIP and PACAP are known

today [6]: VPAC1 and VPAC2 that respond to both VIP and

PACAP and PAC1 that only responds to PACAP. We

observed that the maximum effect of VIP in PCA was

significantly higher compared to both the maximum VIP

response in the coronary arteries and to the maximum

PACAP response in PCA ( p < 0.001, Student’s t-test). At

the same time, there was a trend towards a decreased

maximum response to VIP in PROX compared to LAD.

This was supported by a significantly smaller maximum

response to VIP compared to PACAP in PROX ( p < 0.05,

Student’s t-test). Therefore, our results indicate that VIP

induces different mechanisms of action in the three exa-

mined artery segments. In PCA, VIP may either act through

a new unknown receptor or directly on a different mecha-

nism in the cell membrane, for instance, the potassium

channels. In porcine coronary arteries, it has been demon-

strated that VIP induces relaxation via three mechanisms: a

decrease in [Ca2 +]i by inhibiting the Ca2 + influx (presu-

mably through membrane hyperpolarization mediated by

activation of K+ channels); a decrease in [Ca2 +]i by seques-

tering cytosolic Ca2 + into store sites and a decrease in the

Ca2 + sensitivity of the contractile apparatus through the

activation of G-protein [35]. In an earlier study examining

the vasodilatory effect of VIP on coronary vessels in the

isolated perfused rabbit heart, the slope of the dose–

response curve of VIP suggested that the effect was induced

through one receptor subtype [12]. This is not supported by

the present study, where the Hill-slopes of both proximal

and distal coronary arteries suggest the presence of more

than one receptor subtype.

Alternatively, the regional differences in vascular re-

sponses to the two peptides may be explained by the

interaction of various substances produced and present in

the tissue, or that the vessels display different populations of

the receptor subtypes, some of them being able to counteract

the others or to cluster with each other and G-proteins, and

thereby induce configurational changes. These hypotheses

would explain the observed significant differences for the

Hill coefficients. Earlier, it has been demonstrated in guinea

pig cerebral arteries that the vasoactive mechanism for

neuropeptides may be complex. For instance, NPY, which

is co-localized not only with norepinephrine in sympathetic

perivascular fibres but also with VIP in some parasympa-

thetic neurons, can greatly reduce the vasodilatory effect of

VIP [36]. Recently, it has been demonstrated in rat cerebral

vessels that VPAC1 immunoreactivity is localized to the

plasmalemma of smooth muscle cells on superficial cerebral

arteries and arterioles from the basal surface of the brain

[34]. VIP- and PACAP-containing nerve fibres were found

in separate nerve populations, with different distribution

pattern and density, but both with an intimate contact to the

receptor protein, PACAP to a lesser extent than VIP. In the

present study in rabbits, the number of PACAP-immuno-

reactive nerve fibres in the basal cerebral arteries was higher

than those containing VIP, and VIP seemed to present in a

subpopulation of the PACAP-immunoreactive fibres.

The vasodilatory effect of PACAP in rabbit cerebral

arteries is demonstrated in this study for the first time, but

has previously been reported in other species [37–39].

Earlier studies have demonstrated regional differences in

the vasodilating effect of VIP and PACAP. In canine

cerebral vessels, the maximum vasodilating effect of

PACAP-27 has been demonstrated to be higher, the more

rostrally the vessel is situated [37], with the most potent

vasodilating effect of PACAP-27 in the anterior cerebral

arteries. The vasodilatory potency of VIP and PACAP is

also different in isolated human coronary arteries [14].

Interestingly, it has earlier been demonstrated that the

relative frequency of immunohistochemically visible VIP

T. Dalsgaard et al. / Regulatory Peptides 110 (2003) 179–188186

nerve fibres in cerebral arteries from rabbits was lower than

in several other species, and that the concentration of VIP in

the same arteries was lowest of the examined species,

including cow, pig, cat, guinea pig, rat and mouse [40].

This is the first study examining the effect of clinically

used HRT regimens on the function of non-atherosclerotic

coronary and cerebral arteries, at the same time excluding

the influence of phytoestrogens. Earlier, we have observed

that feeding with a standard plant-component-based diet is

able to change the effect of VIP on atherosclerotic cerebral

arteries (Dalsgaard et al., unpublished data). In the present

study, pretreatment with sex steroids, leading to physiolo-

gical plasma levels of estradiol, and plasma levels of estrone

that confirmed the uptake of NETA, did not influence the

peptide-induced vasodilation. Similar treatment regimens in

female rabbits have previously been reported to induce

significant changes in the functional and mechanical cha-

racteristics of cerebral and coronary arteries [29]. In non-

vascular uterine rabbit smooth muscle, treatment with estra-

diol and progesterone for 4 weeks was able to induce

significant changes in the receptor expression and effect of

VIP [30]. Thus, the cardioprotective effects produced by

estradiol in non-atherosclerotic arteries seem not to involve

the actions of VIP/PACAP directly.

Acknowledgements

This study was supported by grants from the Danish

Medical Research Council (SSVF), the Novo Nordisk

Foundation, the Danish Heart Foundation and the Research

Council of the Copenhagen Hospital Corporation. The

authors also wish to thank Novo Nordisk Farmaka, Lyngby,

Denmark, for supplying 17h-estradiol and Norethindrone

Acetate for the study.

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