International Journal of Impotence Research (2002) 14, 271–282
ß 2002 Nature Publishing Group All rights reserved 0955-9930/02 $25.00
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Morphological and functional characterization of a rat vaginal
smooth muscle sphincter
A Giraldi1,4*, P Alm3, V Werkström2, L Myllymäki2, G Wagner1 and KE Andersson2
1
Division of Sexual Physiology, Department of Medical Physiology, University of Copenhagen, Rigshospitalet,
Copenhagen, Denmark; 2Department of Clinical Pharmacology, Lund University Hospital, Lund, Sweden; 3Department of
Pathology, Lund University Hospital, Lund, Sweden; and 4Department of Clinical Pharmacology, Rigshospitalet,
Copenhagen, Denmark
The aim of the present study was to gain information about adrenergic-, cholinergic- and nonadrenergic, non-cholinergic (NANC)- transmitter systems=mediators in the rat vagina, and to
characterize its smooth muscles functionally. Tissue sections from vagina of Sprague Dawley rats
were immunolabelled with antibodies against protein gene product 9.5 (PGP), synaptophysin
(Syn), tyrosine hydroxylase (TH), vesicular acetylcholine transporter (VAChT), neuropeptide Y
(NPY), nitric oxide synthase (NOS), vasoactive intestinal polypeptide (VIP), calcitonin gene-related
peptide (CGRP) and pituitary adenylate cyclase-activating polypeptide (PACAP). Circularly cut
vaginal smooth muscle preparations from the distal vagina were studied in organ baths. In the
paravaginal tissue, a large number of PGP-, NOS-, TH-, VIP-immunoreactive (IR) and few CGRP-IR
nerve trunks were observed, giving off branches to the smooth muscle wall. The smooth muscle
wall was supplied by a large number of PGP-, Syn-, VAChT-, NPY-, NOS- and TH- IR nerve
terminals, whilst only a moderate to few numbers of CGRP-, VIP- and PACAP-IR terminals were
identified. Especially the distal part of the vaginal wall, where the circularly running smooth
muscle was thickened into a distinct sphincter structure, was very richly innervated,
predominantly by PGP- and NOS-IR terminals. Below and within the basal parts of the epithelium
in the distal half of the vagina, a large number of PGP- and few NOS- and PACAP-IR varicose
terminals were observed. The vaginal arteries were encircled by plexuses of nerve terminals. A
large number of these were PGP-, Syn-, VAChT-, NOS-, TH-, NPY- and VIP-IR, and few were
CGRP- and PACAP-IR. In isolated preparations of the distal vagina, electrical field stimulation
(EFS) caused frequency-dependent contractions, which were reduced by sildenafil, tetrodotoxin
(TTX) and phentolamine. In preparations contracted by norepinephrine (NA), EFS produced
frequency-dependent relaxations. Pretreatment with the NOS-inhibitor NG-nitro-L-arginine, TTX,
or the inhibitor of soluble guanylate cyclase, ODQ, abolished the EFS relaxations. In NE
precontracted preparations, cumulative addition of sildenafil caused concentration-dependent
relaxation. Carbachol contracted the strips concentration-dependently from baseline. It can be
concluded that the distal part of the rat vagina forms a distinct smooth muscle sphincter, which is
richly innervated by adrenergic, cholinergic and NANC nerves. The present studies suggest that in
the rat the L-arginine=NO-system not only plays an important role in the regulation of vaginal
smooth muscle tone, but also affects blood flow, and may have sensory functions.
International Journal of Impotence Research (2002) 14, 271–282. doi:10.1038=sj.ijir.3900886
Keywords: sexual function; female; rat; smooth muscle; vagina; organ bath; immunohistochemistry
Introduction
During the sexual arousal response in females the
physical and psychological stimuli causes lubrication of the vagina, an important step in order to
facilitate intromission.1 Similar to the erectile
*Correspondence: A Giraldi MD, Department of Clinical
Pharmacology, Rigshospitalet, Div. 7642, Blegdamsvej 9, 2100
Copenhagen, Denmark.
E-mail: [email protected]
Received 27 August 2001; accepted 5 March 2002
mechanism in males, this physiological response
in the female may depend on neurally regulated
vaginal and vascular smooth muscle relaxation,
leading to increased vaginal blood flow, vasocongestion and transudation of fluid into the vaginal
lumen.2 Dysfunctions in the neurogenic regulation
may impair the arousal response and lead to female
sexual dysfunction (FSD). However, little is known
about the transmitters regulating the female sexual
arousal response.
Several populations of autonomic nerves innervate the vagina and its vasculature. In several
species, including the human, the transmitters are
Rat vaginal smooth muscle sphincter
A Giraldi et al
272
not only noradrenaline3 and acetylcholine,4 but
include non-adrenergic, non-cholinergic (NANC)transmitters=modulators. Among the putative
NANC transmitters=modulators are vasoactive intestinal polypeptide (VIP),5 – 10 nitric oxide (NO,
produced by NO synthase, NOS),6,10,11 neuropeptide Y (NPY),5,6,12 calcitonin gene-related peptide
(CGRP),6,8 substance P,10 pituitary adenylate
cyclase-activating polypeptides (PACAP),13 – 15 helospectin,13 and peptide histidine methionine
(PHM).5 Very little is known about the role of these
systems in the regulation of the vaginal smooth
muscles.
NO is believed to cause smooth muscle relaxation, and also to modulate the action of other
transmitters by activating soluble guanylate cyclase
leading to a raise in intracellular levels of cyclic
GMP (cGMP). Sildenafil, an inhibitor of type 5
phosphodiesterase (the enzyme responsible for
breakdown of cGMP), has been developed as a
treatment of male erectile dysfunction, due to its
relaxing effect on penile smooth muscle.16
Although the importance of the L-arginine=NO
system in the control of smooth muscle is well
established in the male erectile mechanism17,18 and
in various other regions, including the myometrium,19 the gastrointestinal tract,20 the vascular
system21 and the lower urinary tract,22 little is
known about the significance of this system in the
female genital tract.1,23
The aim of the present study was to investigate
the distribution of nerves containing adrenergic-,
cholinergic-, and NANC- transmitters=modulators in
the rat vagina. In addition, we wanted to establish
an in vitro model for functional studies of vaginal
smooth muscle to characterize adrenergic-, cholinergic- and NANC mechanisms in this tissue, with
focus on the NO system.
Materials and methods
Tissue preparation for acetylcholinesterase (AChE)
histochemistry and immunohistochemistry
Female Sprague Dawley rats (B & K Universal,
Stockholm, Sweden; weighing 250 – 300 g) were
used.
For the demonstration of AChE activity four rats
were killed by CO2 asphyxia. Thereafter the whole
genital tract was dissected out en bloc, and tissue
specimens were taken and frozen immediately in
isopentane at 7 40 C and thereafter stored at
7 70 C.
For immunohistochemistry six rats were perfused
through the left heart ventricle into the aorta. First,
they were perfused with 100 ml ice – cold calciumfree Krebs buffer containing heparin (10 000 IU=l;
International Journal of Impotence Research
Heparin1, Lövens, Malmö, Sweden) and sodium
nitrite (0.5 g=l), and then with 300 ml of an ice – cold,
freshly prepared solution of 4% formaldehyde (FA)
in phosphate buffered saline (PBS; 0.1M, pH 7.4).
Thereafter, the genital tract was dissected out en
bloc and further immersion-fixed for additional 4 h
in the FA=PBS buffer. The tissue specimens were
then rinsed in 15% sucrose in PBS (three rinses
during 48 h) and finally frozen in isopentane at
7 40 C and then stored at 7 70 C.
For both series of experiments, cryostat sections
were cut at a thickness of 8 mm, thaw-mounted onto
glass slides and air-dried for about 2 h (AChEhistochemistry), or for about 30 min (immunohistochemistry), and then processed as described below.
Experimental procedure
Acetylcholinesterase histochemistry. Sections were
processed according to the copper thiocholine
method of Koelle and Friedenwald24 as modified
by Holmstedt.25 After air-drying the sections were
incubated for 6 h in a substrate containing prometazine for the inhibition of non-specific cholinesterase
activity.26 Thereafter the sections were rinsed in
distilled water and counter-stained in eosin.
Immunohistochemistry. Following air-drying the
sections were pre-incubated in PBS with 0.2%
Triton X-100 for about 2 h at room temperature.
Some sections were then incubated overnight at 4 C
with rabbit antisera against protein gene product 9.5
(PGP), NPY, PACAP-27, synaptophysin (Syn), tyrosine hydroxylase (TH), VIP, vesicular acetylcholine
transporter (VAChT), or a 15-amino acid sequence
from the C-terminal part of a cloned rat cerebellar
NOS.27,28 Other sections were incubated overnight
at 4 C in goat antisera to NPY or VAChT, in sheep
antisera to neuronal NOS29 or NPY, or in guinea pig
antisera to CGRP or VIP or a mouse monoclonal
antiserum recognizing both PACAP-27 and PACAP38. After rinsing in PBS (three rinses during 10 min),
the sections were incubated for 90 min with FITC(fluorescein isothiocyanate) conjugated swine antirabbit or donkey anti-goat immunoglobulins (IgG),
or Texas Red (TR)-conjugated donkey anti-goat,
donkey anti-guinea-pig, donkey anti-rabbit, or donkey anti-sheep IgG. After rinsing, the sections were
mounted in PBS=glycerol with p-phenylenediamine
to prevent fluorescence fading.30 For the simultaneous demonstration of two antigens,31 some sections were incubated overnight with an antiserum to
the first primary antigen, rinsed, and then incubated
overnight with an antiserum to the second primary
antigen raised in a different species. After rinsing,
the sections were incubated for 90 min in FITCconjugated IgG to one of the primary antisera,
Rat vaginal smooth muscle sphincter
A Giraldi et al
rinsed, and then incubated for 90 min in TR- or
TRITC- (tetramethyl-rhodamine isothiocyanate)
conjugated IgG to the second primary antiserum.
The sections were then rinsed and mounted as
described earlier.
All primary and secondary antisera used, and
their sources and corresponding working solutions
are listed in Tables 1 and 2. The combinations of
primary and secondary antisera used for the double
immunolabellings are shown in Table 3. All antibodies (primary and secondary) were diluted in
PBS.
All sections were examined with an Olympus
3650 system microscope, equipped with epi-illumination and filter settings for FITC, TRITC and TRimmunofluorescence.32 In control experiments no
immunoreactivity could be detected in sections
incubated in the absence of the primary antisera,
or with antisera absorbed with excess of the
respective antigen (100 mg=ml; except rabbit TH
and sheep NOS for which antigenic substances were
not available).
As cross reactions to antigens sharing similar
amino acid sequences cannot be completely excluded, the structures demonstrated are referred to
as CGRP-, NOS-, NPY-, PACAP-, PGP-, Syn-,
VAChT-, VIP- or TH-IR (immunoreactive). The
immunoreactive structures were evaluated with
respect to type of nerve structures (nerve trunks,
non-varicose nerve fibres, and varicose terminals),
273
Table 3 Combinations of antisera used for double immunolabelling
Combinations of primary
antisera (Table 1)
Combinations of secondary
antisera (Table 2)
NOS (ra) þ NPY (go)
NOS (ra) þ VIP (gp)
NOS (ra) þ CGRP (gp)
NOS (ra) þ PACAP (mo)
VAChT (ra) þ NOS (sh)
VAChT (ra) þ VIP (gp)
VAChT (ra) þ CGRP (gp)
TH (ra) þ VAChT (go)
NPY (ra) þ VAChT (go)
NPY (ra) þ NOS (sh)
Syn (ra) þ VAChT (go)
Syn (ra) þ NOS (sh)
Syn (ra) þ NPY (sh)
Syn (ra) þ VIP (sh)
PACAP (ra) þ VaChT (go)
PACAP (ra) þ NOS (sh)
2þ8
3þ8
3þ8
1 þ 11
1þ9
1 þ 10
1 þ 10
1þ7
1þ7
1þ9
3þ8
5þ8
5þ8
4þ8
1þ7
1þ9
(ra) ¼ rabbit,
(go) ¼ goat,
(sh) ¼ sheep,
(gp) ¼ guinea-pig,
(mo) ¼ mouse.
The primary antisera are described in Table 1. The numbers of the
secondary antisera correspond to those in Table 2.
Table 1 Primary antisera used for immunohistochemical experiments
Antiserum
Host
Working dilution
PGP
NOS
NPY
Syn
VIP
VAChT
VAChT
NOS
VIP
CGRP
TH
NPY
PACAP
PACAP
(ra)
(ra)
(ra)
(ra)
(ra)
(ra)
(go)
(sh)
(gp)
(gp)
(ra)
(go)
(mo)
(ra)
1:2000
1:1280
1:640
1:1000
1:1280
1:2400
1:1600
1:6000
1:640
1:640
1:200
1:800
1:2
1:600
Source
Ultraclone, Wellow, Isle of Wight, UK
Euro-Diagnostica, Malmø, Sweden
Euro-Diagnostica, Malmø, Sweden
Euro-Diagnostica, Malmø, Sweden
Euro-Diagnostica, Malmø, Sweden
Euro-Diagnostica, Malmø, Sweden
Chemicon, Malmø, Sweden
P.Emson, Babraham Institute, Cambridge, UK
Euro-Diagnostica, Malmø, Sweden
Euro-Diagnostica, Malmø, Sweden
Pel-Freez, Rogers, AR, USA
T. Schwartz, Dept. Clin. Chemistry, Rigshospitalet, Copenhagen, DK
J. Hannibal, Dept. Clin. Chemistry, Bispebjerg Hospital, Copenhagen, DK
Peninsula Lab Inc, Belmont, CA, USA
(ra) ¼ rabbit, (go) ¼ goat, (sh) ¼ sheep, (gp) ¼ guinea-pig, (mo) ¼ mouse.
Table 2 Secondary antisera used for immunohistochemistry
Antiserum
Host
1 FITC
2 FITC
3 FITC
4 FITC
5 FITC
6 FITC
7 TR
8 TR
9 TR
10 TR
11 TRITC
Swine anti-rabbit
Donkey anti-goat
Donkey anti-goat
Donkey anti-guinea-pig
Donkey anti-sheep
Goat anti-guinea-pig
Donkey anti-goat
Donkey anti-rabbit
Donkey anti-sheep
Donkey anti-guinea-pig
Goat anti-mouse
Working dilution
1:80
1:80
1:80
1:80
1:80
1:80
1:125
1:125
1:125
1:125
1:40
Code
Source
F 0205
PF 240
705-095-147
706-095-148
713-095-147
F 6261
705-076-147
711-075-152
713-075-147
706-075-148
T 5393
Dakopatts, Stockholm, Sweden
The Binding site, Birmingham, UK
Jackson ImmunoResearch, West Grove,
Jackson ImmunoResearch, West Grove,
Jackson ImmunoResearch, West Grove,
Sigma, St. Louis, MO, USA
Jackson ImmunoResearch, West Grove,
Jackson ImmunoResearch, West Grove,
Jackson ImmunoResearch, West Grove,
Jackson ImmunoResearch, West Grove,
Sigma, St. Louis, MO, USA
PA, USA
PA, USA
PA, USA
PA,
PA,
PA,
PA,
USA
USA
USA
USA
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274
and to number (large, moderate and few). In the
evaluation of the latter parameter, PGP was used as
an internal standard for the total innervation, as PGP
is a cytoplasmic protein that can be used for the
demonstration of all types of nerve fibres.33,34
Synaptophysin is a membrane constituent of presynaptic transmitter vesicles of neurons and can
therefore be used as a marker for varicose neuronal
terminals.35,36
Profiles were considered as coinciding when, in
the double immunolabelling, at least three varicosities and intervaricose segments were identical.
(1 – 40 Hz) were recorded in the absence and
presence of the NO-synthase inhibitor N-nitro-Larginine (L-NOARG; 1074 M), the inhibitor of
soluble guanylate cyclase, 1H-[1,2,4] oxidiazolo
[4,3-a] quinoxalin-1-one, ODQ (1075 M), TTX
(1076 M) or the type 5 phosphodiesterase inhibitor, sildenafil (361076 M).
In NA precontracted tissue strips, the relaxant
effect of sildenafil was recorded by cumulative
addition of sildenafil (1078 M – 361075 M).
NA-precontracted (361076 M) tissue strips and
strips at basal tone were exposed to increasing
concentrations of carbachol (1078 – 1074 M)
Tissue preparation for organ bath experiments
Determination of oestrous cycles
Female rats (Sprague Dawley, weight 200 – 250 g)
were killed by CO2 asphyxia. After killing of the
animals, a vaginal smear was done in order to
determine the oestrous cycles of the animals (see
description later). Thereafter the abdomen was
opened and the uterus, bladder and vagina were
removed en bloc and placed in a chilled Krebs
Ringer solution (for composition see later). The
vagina was cut open and vaginal strips
(16167 mm) were dissected out. Strips cut in
longitudinal and circular directions were prepared.
Vaginal smears were coated on glass slides by using
cotton buds moistened in 0.9% saline, The smears
were fixed in 70% ethanol and stained with
haematoxylin and erythrocine. The oestrous phase
was determined blinded from morphological characteristics of the smears. The actual oestrous phase
of each animal was unknown to the investigator
when the experiments were performed.
Drugs and solutions
Recording of mechanical activity
The strips were transferred to 5 ml temperature
regulated (37 C) organ baths containing Krebs solution continuously bubbled with a mixture of 95% O2
and 5% CO2 to keep the pH at 7.4. Registration of
isometric tension and electric field stimulation,
were performed as described previously.37 After
mounting, the tissue strips were stretched to a
passive tension of 4 – 5 mN and allowed to equilibrate for 60 min. To assess the contractile capacity of
the tissue strips, each experiment was started by
exposing the preparation to a high Kþ-solution (for
composition see later).
The following drugs were used: N-nitro-L-arginine,
phentolamine, noradrenaline, tetrodotoxin, carbamylcholine chloride (Sigma Chemical Company, St
Louis, MO, USA), 1H-[1,2,4] oxidiazolo [4,3-a]
quinoxalin-1-one (ODQ; Tocris Cookson Ltd, Bristol,
UK) and sildenafil citrate (Pfizer, Sandwich, UK).
The Krebs solution had the following composition (mM): NaCl 119, KCl 4.6, CaCl2 1.5, MgCl2 1.2,
NaHCO3 15, NaH2PO4 1.2, glucose 5.5 High Kþsolution (124 mM) was prepared by replacing NaCl
with equimolar KCI.
Analysis of data
Experimental protocol
The following experiments were then performed:
The frequency-dependent contractile responses
to electrical field stimulation (EFS) (1 – 50 Hz)
were recorded. Thereafter the effect of phentolamine (1076 M), tetrodotoxin (TTX, 1076 M) and
increasing doses of sildenafil (1078 M361075 M) on single frequency EFS (20 –
30 Hz) contractions was investigated.
In norepinephrine (NA) (361076 M) precontracted tissue, the relaxant responses to EFS
International Journal of Impotence Research
Contractile effect of EFS and carbachol has been
expressed as percentage of the tension elicited by
high Kþ (124 mM). Relaxant effects of agonists on
EFS contractions were expressed as percentage
reduction of maximum contraction prior to addition
of agonist. Agonist relaxation of NA precontracted
preparations was expressed as percentage reduction
of the NA-induced tension. n Denotes the number of
animals and strip preparations. The values are given
as means s.e. of the mean and statistical analysis
were performed by Student’s two-tailed t-test. A
probability of P < 0.05 was considered as significant.
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A Giraldi et al
Results
Immunohistochemistry and acetylcholine esterase
staining
Methodology. No overt differences in number and
distribution could be observed regarding nerve
terminals expressing AChE-positive reaction and
VAChT-immunoreactivity, the latter being a more
specific marker for the delineation of cholinergic
nerve terminals.38
IR terminals were significantly less abundant (few to
moderate number).
The smooth muscle layer was thin in the upper
part of the vagina, whereas in the distal part the
thickness of the smooth muscle layer increased.
Around the vaginal orifice, the circular smooth
musculature was thickened into a distinct sphincter
structure (Figure 2b). In the sphincter, the number of
PGP- and NOS-IR varicose terminals was large
(Figure 3a and b), whereas that for VAChT- and
TH-IR nerve terminals was moderate (Figure 3c and
d).
275
Paravaginal tissue. In the paravaginal connective
tissue, coarse PGP-, NOS-, TH- and VIP-IR and
AChE-positive nerve trunks were observed running
along the vaginal wall. A large number of PGP- and
NOS-IR nerve trunks were identified. They divided
into less coarse branches, which followed the
bundles of the smooth muscle layer. In a few nerve
trunks single CGRP-IR fibres were noticed. Furthermore, few scattered clusters of PGP- and NOS-IR
ganglions with neuronal cell bodies, from which
coarse nerve trunks were derived, were observed
(Figure 1). In contrast, no Syn-, VAChT-, NPY- or
PACAP-IR nerve trunks could be found.
Smooth muscle wall. PGP-, Syn-, VAChT-, NOS-,
NPY-, CGRP-, TH-, PACAP- and VIP-IR and AChEpositive nerve terminals were shown to supply the
smooth muscle bundles constituting the vaginal
wall (Figure 2a). The innervation was most pronounced in the distal part of the vagina. PGP- and
Syn-IR terminals were most frequent (large number),
whereas VAChT-, NOS-, NPY- and TH-IR, and
AChE-positive nerve terminals were less numerous
(moderate). In comparison, VIP-PACAP- and CGRP-
Figure 1 Ganglion with NOS-IR cell bodies in paravaginal
connective tissue. Bar ¼ 100 mm.
Figure 2 (a) Single smooth muscle bundles (arrows) of the
vaginal wall. (b) Sphincter of smooth muscle bundles (arrows)
surrounding the vaginal orifice. Compare with the thin smooth
muscle layer in (a). Note that the grade of magnification is almost
the same in (a) and (b). Masson Trichrome staining.
Bars ¼ 100 mm.
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276
Figure 3 Smooth muscle sphincter surrounding the vaginal orifice, with various types of nerve terminals running along the bundles of
smooth muscle cells. (a) PGP-IR terminals, (b) NOS-IR terminals, (c) VAChT-IR terminals, (d) TH-IR terminals. Bar ¼ 50 mm.
Mucosa. In the distal half of the vagina, immediately below the epithelium, a large number of PGPand few NOS- and PACAP-IR varicose terminals
were observed. They gave off branches that ran into
the epithelium towards the epithelial surface being
clearly more gracile and displaying a thinner
diameter than PGP-IR nerve terminals in other
structures (Figure 4a and b). In contrast, no
epithelium-related Syn-, VAChT-, VIP-, NPY-, THand CGRP-IR and AChE-positive terminals could be
demonstrated. Furthermore, no nerve fibres of any
category related to the epithelium could be detected
in the proximal half of the vagina.
Arteries. The most conspicuous finding was that of
plexuses of nerve terminals encircling vaginal
arteries of various sizes. A large number of these
plexuses were VAChT-, NOS-, VIP-, TH-, NPY-,
PGP- and Syn-IR and AChE-positive, whilst a few
CGRP- and PACAP-IR were demonstrated (Figure 5a
and b).
Double immunolabelling
In the plexuses related to the arteries and the smooth
muscles of the vaginal wall, coinciding profiles were
International Journal of Impotence Research
Figure 4 (a) Intraepithelial PGP-IR terminals, and (b) a single
intraepithelial NOS-IR terminal directed towards the epithelial
surfaces (right). Bar ¼ 100 mm.
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A Giraldi et al
Figure 5 Paravaginal artery surrounded by a plexus of VAChT(a) and NOS-IR (b) terminals, which to a large extent show
coinciding profiles (arrow heads). Double labelling of the same
section in (a) and (b). Bar ¼ 100 mm.
observed between VAChT-and NOS-, VAChT- and
VIP-, NOS- and PACAP-, and VAChT- and PACAPIR terminals (see Figure 6a – h) as well as between
NOS- and VIP-IR terminals. In comparison, less than
half of the VAChT- or NOS-IR terminals showed
coinciding profiles with NPY-IR terminals indicating that most of the NPY-IR terminals were VAChTor NOS-negative (Figure 6k and l). It was not possible
to demonstrate differences in patterns of double
labelling between different regions of the vagina.
In none of the smooth muscle and vascular
related terminals, did CGRP-IR terminals coincide
with terminals expressing VAChT- or NOS-immunoreactivity, although their profiles sometimes
appeared to be very similar.
induced contraction. In the following experiments,
the tissue strips were contracted by single frequency
EFS (n ¼ 11). These contractions were inhibited by
sildenafil (1078 M – 361075 M) in a concentrationdependent manner as shown in Figure 8. Sildenafil,
361075 M, reduced the contraction to 9% 3% of
the response before addition of sildenafil. Addition
of phentolamine (1076 M, n ¼ 6) reduced the EFSinduced contractions to 3% 2% of the initial level,
and TTX (1076 M, n ¼ 6) abolished the contractions
(data not shown). In the NA precontracted tissue
(n ¼ 7), EFS evoked frequency-dependent relaxations as shown in Figure 9. Maximal relaxation
was induced at 30 Hz leading to 31 5% reduction
of the NA-induced contraction. Incubation with LNOARG (1074 M) (Figure 9a), ODQ (1075 M) (Figure
9b) or TTX (1076 M) (data not shown) abolished
these relaxations, and in the ODQ experiments,
contractions were shown at high frequency
stimulation.
Addition of sildenafil (361075 M, n ¼ 7) to the
NA precontracted preparations followed by EFS,
showed that sildenafil provoked increased as well as
longer lasting EFS-induced relaxations (Figure 10a &
b). In the sildenafil treated preparations, maximal
relaxation was increased to 57 8% (n ¼ 7) of the
precontraction, which was significantly (P < 0.05)
higher than in controls.
In NA-precontracted tissue, sildenafil (1078 M –
361075 M, n ¼ 5) relaxed the tissue concentrationdependently with maximal relaxation at 361075 M
(Figure 11). Carbachol (1078 – 1074 M, n ¼ 8) contracted the vaginal tissue concentration-dependently from baseline (Figure 12), whilst it had no
effect upon NA-precontracted tissue strips (data not
shown).
In both the immunohistochemical and functional
studies we were not able to demonstrate any effect of
the oestrous cycle upon the results. The animals
were equally distributed between the four cycles:
oestrous, pro-oestrous, metoestrous and dioestrous.
However, the number of animals is small and large
scale studies have to be done in order to establish a
possible measurable effect of the oestrous phase
upon morphological and functional parameters.
277
Functional studies
Discussion
Only weak responses to Kþ (124 mM), and no or
weak responses to EFS and NA could be evoked in
circular proximal strips and longitudinal strips from
the rat vagina. Strips cut in circular direction from
the distal vagina showed reproducible contractile
responses and were used for further studies.
EFS (1 – 50 Hz) evoked frequency-dependent contractions of the vaginal tissue as shown in Figure 7.
The contractions reached maximum at 40 Hz, and
the strips reached a contraction level that represented 85% 9% (n ¼ 11) of the 124 mM Kþ-
The present study showed that the rat vagina has a
rich innervation in many of its structures, as seen
from prominent labelling by the neuronal markers
PGP and Syn, of which PGP labels all types of nerve
fibres whereas Syn specifically labels synaptic
terminals. In the smooth muscle layers a large
number of Syn-, NOS-, NPY-, TH- and VAChT-IR
and AChE-positive nerve terminals were identified,
whilst CGRP-, PACAP- and VIP-IR terminals were
few to moderate in number. These observations
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278
Figure 6 Coinciding profiles between various types of nerve terminals (arrow heads) in smooth muscle bundles of the sphincter
surrounding the vaginal orifice (a – h), and a paravaginal vascular plexus (k – l). Pairs of the same, double immunolabelled sections in the
left and right panels. (a, b) Double labelling for VAChT- and NOS-IR terminals, (c, d) Double labelling for VAChT- and VIP-IR terminals,
(e, f) Double labelling for VAChT- and PACAP-IR terminals, (g, h) Double labelling for NOS- and PACAP-IR terminals, (k – l) Double
labelling for VAChT- and NPY-IR terminals. Bars ¼ 100 mm.
International Journal of Impotence Research
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279
Figure 7 Original tracing demonstrating the frequency-dependent contractile effect of electrical field stimulation (1 – 50 Hz) of
distal vaginal smooth muscle.
Figure 8 Original tracing demonstrating the inhibitory effect of
sildenafil on electrical field stimulated (30 Hz) contractions in
distal vaginal smooth muscle.
Figure 10 (a, b) Effects of sildenafil (361075 M) on electrically
evoked relaxations (1 – 40 Hz) in NA-contracted distal vaginal
smooth muscle. (a) Original tracing demonstrating the enhancing
effect of sildenafil on electrical field stimulated relaxations. (b)
Graph displaying relaxations evoked by electrical field stimulation, before (open circles) and after (closed circles) addition of
sildenafil. Values represent means standard errors of the means
(n ¼ 7, *P < 0.05; **P < 0.01).
Figure 9 (a, b) Original tracing demonstrating relaxations evoked
by electrical field stimulation (1 – 40 Hz) in distal vaginal smooth
muscle, before and after addition of (a) N-nitro-L-arginine (LNOARG, 1074 M) or (b) 1H-[1,2,4] oxidiazolo [4,3-a] quinoxalin-1one (ODQ, 1075 M).
could mean that the vaginal smooth muscle cells are
regulated, by adrenergic, cholinergic and NANC
mediators. This is in good accordance with other
studies of the vagina from rats and other species,
although the density of the different transmitters
may differ from study to study and among species.3,4,7,8,11
A finding in the present study, which to the best
of our knowledge, has not been described earlier,
was the morphological identification of a sphincter
structure in the distal part of the vagina. This was
seen as a thickening of the circular smooth musculature, compared to other parts of the vagina in
which the smooth muscle layers were considerably
thinner. The described smooth muscle sphincter
structure differs from the bulbocavernosus muscle,
usually known as sphincter vaginae.39
Figure 11 Graph displaying the relaxing effect of sildenafil
(1078-361075 M) on NA-contracted distal vaginal preparations.
Values represent means standard errors of the means (n ¼ 5).
The smooth muscle sphincter structure contained
a pronounced innervation of various populations of
nerves, especially of NOS-IR nerves. Also in the rat
urethra a sphincter-like smooth muscle structure
can be found, which is richly innervated by a large
number of adrenergic, cholinergic and NPY and
NOS containing nerves, and fewer VIP- and CGRPIR nerves,40 findings similar to the present observations in rat vagina. Further, in the rat urethra
functional data have confirmed the morphological
findings of a significant role for NO in the regulation
of urethral smooth muscle functions.22 Thus, the
International Journal of Impotence Research
Rat vaginal smooth muscle sphincter
A Giraldi et al
280
Figure 12 Concentration – response curve obtained by cumulative addition of carbachol to isolated distal vaginal smooth
muscle preparations. Each point is expressed as percentage of
contraction induced by 124 mM Kþ. Values represent means standard errors of the means (n ¼ 8).
large number of NOS containing nerves in the
vaginal sphincter structure suggests an important
role of the NO system in regulating smooth muscle
functions also in the rat vagina.
We have also shown that the subepithelial
innervation differed between different parts of the
vagina, with an intraepithelial innervation of the
distal vagina by a large number of PGP-IR terminals.
These were clearly more gracile and had a smaller
diameter than PGP-IR terminals in other structures.
Most probably, these nerve terminals represent Cfibres with sensory functions, ie for receiving and
propagating tactile stimuli in the sexual arousal
response. Some of these intraepithelial nerve terminals were PACAP-IR as previously described, and
are proposed to have a sensory function.15 Other
intraepithelial nerve terminals were NOS-IR, and
could be derived from NOS-IR cell bodies in dorsal
root ganglia.41,42 Since these NOS-IR fibres were not
linked anatomically to any motor function, it could
be assumed that besides regulating smooth muscle
function and blood flow, another important function
of NO in the vagina might be sensory. However, a
possible sensory function involving NO has to be
established by further investigation.
The present study also demonstrated plexuses of
various types of terminals encircling the vaginal
arteries. These findings suggest a role of adrenergic-,
cholinergic- and NANC-mediators in the regulation
of vaginal blood flow, and thereby vaginal lubrication. In a rat in vivo model Giuliano et al have
demonstrated that stimulation of the sacral parasympathetic outflow and of the hypothalamus
mimics the physiological response of the vagina
during sexual arousal, and that stimulation of the
sympathetic chain opposes this response.43
International Journal of Impotence Research
The general role of NO and VIP as vasodilators is
well known.21,44 In humans, VIP is considered as an
important neurotransmitter for mediating vaginal
vasodilatation and thereby lubrication during sexual
arousal.45 In humans and rats, PACAP is considered
to be an important vasodilator in the genital
tract.14,46 In rabbits, Min et al demonstrated that
sildenafil increases vaginal blood flow induced
by pelvic nerve stimulation.47 In humans, Hoyle
et al also demonstrated innervation of vaginal
arteries by CGRP-, NPY-, VIP-IR nerve fibres implying a role of these transmitters in the regulation of
vaginal blood flow.6 However, further study of
transmitters in regulation of vaginal blood flow is
needed.
The co-localization studies revealed that in the
nerves innervating the smooth muscles of the
vaginal wall and the vasculature, NOS, VAChT,
VIP and PACAP are co-localized. Previous studies
have demonstrated co-localization of PACAP and
VIP in the human vaginal smooth muscle43 and of
NOS and VIP in the perivascular and smooth muscle
varicose terminals in the porcine vagina.10 The
functional significance of the demonstrated colocalizations has not been established. Very likely,
NO could interact with VIP and PACAP in the
regulation of the inhibitory tone of vaginal smooth
muscle as previously suggested in other smooth
muscle tissues.32,48,49 Further, the present intraneuronal co-localization of VAChT and NOS immunoreactivities does not also exclude the possibility of an
influence of NO on excitatory cholinergic neurotransmission. This has been suggested in colonic
smooth muscle, in which NO might have a modulatory function on presynaptic release of acetylcholine, or exert a postjunctional inhibitory role on
cholinergic transmission.50,51
Only circularly cut tissue strips from the ‘sphincter-like’ distal structure of the vagina were functional in the organ bath. More proximal — by
location, and longitudinally cut strips did not
respond to stimulation with Kþ and EFS. This lack
of response may reflect a too low amount of
functional smooth muscle cells in the preparations
from the upper part of the vagina, making it
impossible to obtain a measurable response in our
organ bath model. Another reason might be differences in innervation=receptors for transmitters or a
combination of differences in smooth muscle
amount and transmitter systems. The present results
are partly supported by findings in the rabbit vagina
by Berman et al,52 showing that the contractile
ability of the smooth muscle cells was most
pronounced in tissue from the distal part of the
organ. The observed morphological and functional
differences between the upper and lower part of the
vagina may reflect the different embryological origin
of the upper third and lower two-thirds of the
vagina53 or different functional roles of the different
parts, and need further investigation.
Rat vaginal smooth muscle sphincter
A Giraldi et al
The present study demonstrated that NE and
carbachol contracted the distal vaginal smooth
muscle. Sildenafil relaxed the NE precontracted
tissue concentration-dependently. EFS induced
nerve-mediated, a-adrenoceptor dependent contractions, which were inhibited dose-dependently by
sildenafil. We also showed that EFS elicits NANC
relaxations of the vaginal smooth muscle tissue,
which were mediated by the nitrergic system, since
they were blocked completely by the NOS-inhibitor
L-NOARG and enhanced by sildenafil, which is
known to enhance the NO-response. In addition, the
EFS-induced relaxations were blocked by ODQ,
confirming that the cGMP pathway is involved in
these relaxations. These results confirm previous
data from our group, suggesting that NO plays a
major role in regulation of rat vaginal smooth
muscle.37 This is similar to findings in other
urogenital smooth muscles, including clitoral,54
penile,55 lower urinary tract49 and prostate48 smooth
muscle. In human vaginal smooth muscle, Hoyle
et al have demonstrated NOS-IR nerve bundles
innervating the vaginal wall, suggesting a role of
NO in the regulation of vaginal smooth muscle.6
These observations are in good accordance with our
observations on rat vaginal smooth muscle. However, the human tissue specimens may be derived
from other parts of the vagina than the rat tissue
used in the present study, which makes a comparison difficult. Nevertheless, the rat model seems to
be useful in obtaining basic knowledge about
transmitter systems of importance for the vagina
and to generate ideas for studies on human material.
In conclusion, the present results demonstrate a
rich innervation of a rat vaginal sphincter, with
nerves containing adrenergic, cholinergic and
NANC transmitters=mediators. Many nerves contain
more than one transmitter, eg NOS, VIP, VAChT and
PACAP, suggesting that these transmitters could act
in concert in the control of vaginal smooth muscle
activity. Furthermore, the results indicate that the Larginine-=NO-system plays an important role in the
regulation of rat vaginal smooth muscle, as it has
been shown to do in most urogenital smooth muscle
organs. In addition, the L-arginine-=NO-system may
also have important roles in regulation of vaginal
blood flow and in exerting sensory functions.
Identification of the neurotransmitters involved
in the female sexual arousal response gives increased knowledge about mechanisms responsible
for vaginal lubrication and relaxation. Based on this
knowledge future pharmacological treatments of
female sexual dysfunction may be developed.
Acknowledgements
This work was supported by the Swedish Medical
Research Council (grant numbers 11205 and 6837)
and by Pfizer, DK. The authors are grateful to
Lillemor Thuresson, Brita Sunden-Andersson and
Agneta Kristensen, for technical assistance and Dr
Anders Nylen, Department of Clinical Pharmacology, Lund for help with the oestrus cycle studies.
The generous supplies of goat NPY and mouse
monoclonal PACAP antisera from Professor Thue
Schwartz and Dr Jens Hannibal, Departments of
Clinical Chemistry, Rigshospitalet and Bispebjerg
Hospital, Copenhagen, Denmark are greatly appreciated.
281
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