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Eects of vasopressin on the sympathetic contraction of rabbit ear

artery during cooling


1
A.L. Garca-Villalo n,
2
J. Padilla,
1
L. Monge,
1
N. Ferna ndez,
1
M.A. Sa nchez,
1
B. Go mez &
*
,1
G. Die guez
1
Departamento de Fisiolog a, Facultad de Medicina, Universidad Auto noma, Arzobispo Morcillo 2, 28029 Madrid, Spain; and
2
Departamento de Biolog a, Universidad del Atla ntico, Barranquilla, Colombia
1 In order to analyse the eects of arginine-vasopressin on the vascular contraction to
sympathetic nerve stimulation during cooling, the isometric response of isolated, 2-mm segments of
the rabbit central ear (cutaneous) artery to electrical eld stimulation (1 8 Hz) was recorded at 37
and 308C.
2 Electrical stimulation (378C) produced frequency-dependent arterial contraction, which was
reduced at 308C and potentiated by vasopressin (10 pM, 100 pM and 1 nM). This potentiation was
greater at 30 than at 378C and was abolished at both temperatures by the antagonist of vasopressin
V
1
receptors d(CH
2
)
5
Tyr(Me)AVP (100 nM). Desmopressin (1 mM) did not aect the response to
electrical stimulation.
3 At 378C, the vasopressin-induced potentiation was abolished by the purinoceptor antagonist
PPADS (30 mM), increased by phentolamine (1 mM) or prazosin (1 mM) and not modied by
yohimbine (1 mM), whilst at 308C, the potentiation was reduced by phentolamine, yohimbine or
PPADS, and was not modied by prazosin.
4 The Ca
2+
-channel blockers, verapamil (10 mM) and NiCl
2
(1 mM), abolished the potentiating
eects of vasopressin at 378C whilst verapamil reduced and NiCl
2
abolished this potentiation at
308C. The inhibitor of nitric oxide synthesis, L-NOARG (100 mM), or endothelium removal did not
modify the potentiation by vasopressin at 37 and 308C.
5 Vasopressin also increased the arterial contraction to the a
2
-adrenoceptor agonist BHT-920
(10 mM) and to ATP (2 mM) at 30 and 378C, but it did not modify the contraction to noradrenaline
(1 mM) at either temperature.
6 These results suggest that in cutaneous (ear) arteries, vasopressin potentiaties sympathetic
vasoconstriction to a greater extent at 30 than at 378C by activating vasopressin V
1
receptors and
Ca
2+
channels at both temperatures. At 378C, the potentiation appears related to activation of the
purinoceptor component and, at 308C, to activation of both purinoceptor and a
2
-adrenoceptor
components of the sympathetic response.
Keywords: Cutaneous arteries; temperature; vasopressin V
1
receptors; alpha-adrenoceptor vasoconstriction; purinoceptor
vasoconstriction; nitric oxide; endothelium; Ca
2+
-channels; cooling
Abbreviations: BHT-920, 5-allyl-2-amino-5,6,7,8-tetrahydro-4H-thiazolo-[4,5]-dazepin hydrochloride; d(CH
2
)
5
Tyr(Me)AVP,
b-Mercapto-b,b-cyclopenta-methylenepropionyl
1
,O-Me-Tyr
2
,Arg
8
)-vasopressin; L-NOARG, L-N
G
-nitro-arginine;
PPADS, pyridoxalphosphate-6-azophenyl-2-4'-disulphonic acid
Introduction
Vasopressin is known to exert powerful vasoconstrictor
activity in a variety of vascular beds (Altura & Altura, 1977).
However, a key issue is whether the vasoconstrictor action of
vasopressin can be achieved with vasopressin concentrations
that are physiologically or pathophysiologically achievable, as
many studies of the vascular actions of vasopressin have
employed large, unphysiological doses of this hormone (Share,
1988). It has been shown that vasopressin, in addition to its
direct vasoconstrictor eect, may enhance the vascular
response to exogenous catecholamines at low, subthreshold,
doses of this peptide (Bartlestone et al., 1967), although others
have not conrmed this phenomenon (Altura & Altura, 1977).
Although this subject may be of interest for understanding the
role of vasopressin in controlling vascular function, it has
received little attention from investigators.
In a previous study (Padilla et al., 1997), we found that
vasopressin potentiated the vasoconstrictor response of the
rabbit central ear artery, a cutaneous blood vessel, to
sympathetic stimulation at 30 but not at 378C. We therefore
suggested that vasopressin may play a role in the regulation
of the cutaneous circulation during changes in temperature,
by enhancing the sympathetic nerve-induced contraction that
occurs during cooling in cutaneous blood vessels. The
objective of the present study was to analyse further this
potentiating action of vasopressin. Vasopressin receptors have
been classied into V
1
and V
2
subtypes, and in a previous
study we have found that the constriction in several types of
arteries from the rabbit is mediated by the V
1
subtype and
may be modulated by endothelial nitric oxide (Garc a-
Villalo n, 1996). Also, the vasoconstriction to vasopressin
may be produced by mobilization of intracellular Ca
2+
(McDonald et al., 1994) and/or by facilitating the entry of
extracellular Ca
2+
through membrane channels (Altura &
Altura, 1977). Therefore, in the present study we have
analysed the subtype of vasopressin receptor mediating the
potentiating eects of vasopressin on sympathetic contrac-
tion, and have determined whether this potentiating eect is
* Author for correspondence; E-mail: godofredo.dieguez@uam.es
British Journal of Pharmacology (1999) 126, 785 793 1999 Stockton Press All rights reserved 0007 1188/99 $12.00
http://www.stockton-press.co.uk/bjp
dependent on Ca
2+
channels or is modulated by the
endothelium and nitric oxide.
It has been previously reported that sympathetic vasocon-
striction in rabbit ear arteries may be mediated by release of
noradrenaline and ATP from perivascular nerve terminals
(Kennedy et al., 1986). These transmitters then produce
constriction by acting on postjunctional a-adrenoceptors and
purinoceptors, respectively. In the present study we have also
analysed whether the potentiation by vasopressin of the
sympathetic contraction is mediated by a-adrenoceptors and
purinoceptors.
The present studies have been performed at 37 and 308C in
the rabbit central ear artery, a supercial, cutaneous blood
vessel which is involved in thermoregulation (Harker &
Vanhoutte, 1988; Patton & Wallace, 1978; Roberts &
Zygmunt, 1984).
Methods
Thirty-eight New Zealand White rabbits, weighing 2 2.5 kg,
were killed by intravenous injection of sodium pentobarbital
(100 mg kg
71
). Central ear arteries were dissected free and
cut into cylindrical segments 2 mm in length. Each segment
was prepared for isometric tension recording in a 6-ml organ
bath containing modied Krebs-Henseleit solution with the
following composition (millimolar): NaCl, 115; KCl, 4.6;
KH
2
PO
4
, 1.2; MgSO
4
, 1.2; CaCl
2
, 2.5; NaHCO
3
, 25; glucose,
11.1. The solution was equilibrated with 95% oxygen and
5% carbon dioxide to give a pH of 7.3 7.4, which was
measured with a pH-meter micropH 2001 (Crison Instru-
ments). Briey, the method consists of passing two ne,
stainless steel pins, 150 mm in diameter, through the lumen
of the vascular segment. One pin is xed to the organ bath
wall, while the other is connected to a strain gauge for
isometric tension recording, thus permitting the application
of passive tension in a plane perpendicular to the long axis
of the vascular cylinder. The recording system included a
Universal Transducing Cell UC3 (Statham Instruments,
Inc.), a Statham Microscale Accessory UL5 (Statham
Instruments, Inc.) and a Beckman Type RS Recorder
(model R-411, Beckman Instruments, Inc.). A previously
determined resting passive tension of 0.5 g was applied to
the vascular segments, and then they were allowed to
equilibrate for 60 90 min before any drug was added. The
temperature of the bath was adjusted from the beginning of
the experiment at 37 and 308C, and the arteries remained at
the chosen temperature throughout the duration of the
experiment.
Electrical eld stimulation (1, 2, 4 and 8 Hz, 0.2 ms pulse
duration, at a supramaximal voltage of 70 V, during 5 s)
was applied to the arteries with two platinum electrodes
placed on either side of the artery and connected to a CS-
14 stimulator (Cibertec). An interval of at least 5 min was
imposed between stimulation periods to allow recovery of
the response, and the stimulation trains were repeated until
the responses were reproducible over at least 40 min under
control conditions. Thereafter, the eect of vasopressin
(10 pM, 100 pM and 1 nM) on the arterial response to
electrical stimulation was studied by cumulative addition of
this peptide to the organ bath. The segments were
incubated with each vasopressin concentration for 5 min
before electrical stimulation. One stimulation series (1
8 Hz) was applied to the arteries after each dose of the
peptide. Each arterial segment was treated with every dose
of vasopressin. The eect of vasopressin on the response to
electrical stimulation was studied in arterial segments at 37
or 308C. Each arterial segment was tested at one
temperature only.
To investigate the nature of the vasopressin receptor
subtypes involved, the eect of vasopressin on the arterial
response to electrical stimulation was recorded in the presence
of the V
1
vasopressin antagonist, d(CH
2
)
5
Tyr(Me)AVP
(100 nM). In addition, the eect of the V
2
agonist,
desmopressin (1 mM) on the response to electrical stimulation
was studied.
The relative contribution of the sympathetic neurotrans-
mitters noradrenaline and ATP in the eect of vasopressin on
the arterial response to electrical stimulation was studied by
performing a series of experiments in the presence of the a
1
-
and a
2
-adrenoceptor antagonist, phentolamine (1 mM), of the
a
1
-adrenoceptor antagonist, prazosin (1 mM), of the a
2
-
adrenoceptor antagonist, yohimbine (1 mM), of the purinocep-
tor antagonist, pyridoxalphosphate-6-azophenyl-2,4'-disul-
phonic acid (PPADS, 30 mM), and of phentolamine (1 mM)
plus PPADS (30 mM).
The eect of vasopressin on the response to electrical
stimulation was also recorded in the presence of NiCl
2
(1 mM)
and verapamil (10 mM), which are non-specic (Narahashi et
al., 1987) and L-type-specic (McDonald et al., 1994) Ca
2+
-
channel blockers, respectively.
To analyse the role of nitric oxide and the vascular
endothelium, the eects of vasopressin on the response of the
artery to electrical stimulation at 37 and at 308C were studied
in arteries without endothelium or in arteries with endothelium
pretreated with the inhibitor of nitric oxide synthesis L-N
G
-
nitro-arginine (L-NOARG, 100 mM). Endothelium removal
was accomplished by gentle rubbing of the vascular lumen with
a steel rod, and tested, after nishing the experiment with
vasopressin and electrical stimulation, by the abolition of the
relaxing response to acetylcholine (10 mM) after precontraction
with endothelin-1 (100 nM). This peptide was used to
precontract the arteries as in our experimental conditions it
produces more stable contractions than other vasoactive drugs
such as noradrenaline or serotonin.
Reproducible responses to electrical stimulation (1 8 Hz)
were obtained over a period of 40 min. Thereafter, an
antagonist was added to the organ bath, and two series of
electrical stimulation (1 8 Hz) were applied in the presence of
the antagonist. After these series of electrical stimulations,
vasopressin (10 pM, 100 pM and 1 nM) was added to the organ
bath cumulatively and the response to electrical stimulation
was recorded in the arteries in the presence of each
concentration of vasopressin plus the antagonist previously
applied. Each of the antagonists used was added to the bath
40 min before applying vasopressin. As a control, one vascular
segment (each temperature) was treated with vasopressin but
not antagonist.
To examine the site of the eect of vasopressin (i.e., pre- or
post-junctional) on the arterial response to electrical
stimulation, the response of ear arteries to exogenous
noradrenaline, ATP or to the a
2
-adrenoceptor agonist BHT-
920 were studied at 37 or 308C, in the absence (control) and
in the presence of vasopressin (10 pM, 100 pM and 1 nM). The
response to BHT-920 was always studied in the presence of
prazosin (1 mM) to block a possible a
1
agonist eect of this
agonist. A single submaximal concentration (Garc a-Villalo n
et al., 1997b) of noradrenaline (1 mM), BHT-920 (10 mM) or
ATP (2 mM) was used, and it was applied to the arteries
every 15 min, each time followed by washing, until a
consistent response was obtained over at least 40 min. After
Vasopressin and sympathetic response 786 A.L. Garc a-Villalo n et al
this period, vasopressin, in increasing concentrations (10 pM,
100 pM and 1 nM), was added to the bath, and 15 min
thereafter noradrenaline, BHT-920 or ATP were again
applied to the arteries, followed by washout and addition of
the next vasopressin concentration.
Data are expressed as means+s.e.mean, and were evaluated
by analysis of variance (ANOVA) applied to each group of
data. To compare the response in the presence and the absence
of vasopressin, paired Student's t-test after analysis of variance
was applied to the absolute contraction values at each
temperature. Then, to compare the eects of vasopressin
found at 37 and 308C, the increments or decrements in the
contraction of arteries to sympathetic stimulation were
calculated (i.e., the dierence between the control response
and the response in the presence of vasopressin) and three-way
analysis of variance was applied to these data: in this case one
factor was stimulation frequency, another factor was
vasopressin concentration and another was temperature. To
analyse the eects of the antagonists on the potentiation by
vasopressin, the increments or decrements in the contraction
produced by electrical stimulation were also calculated and
analysed by three-way analysis of variance, in which one factor
Figure 1 Contraction of rabbit ear arteries at 37 and 308C to electrical eld stimulation (1 8 Hz, 0.2 ms pulse duration, 70 V, for
5 s) in arteries: (a) in the absence (control) and in the presence of vasopressin (10 pM 1 nM); (b) pretreated with the antagonist of
vasopressin V
1
receptors d(CH
2
)
5
Tyr(Me)AVP (100 nM) in the absence (control) and in the presence of vasopressin (10 pM 1 nM);
and (c) in the absence (control) and in the presence of desmopressin (1 mM). Points are means+s.e.mean. * Signicantly dierent
(P50.01) from the control. n=number of animals.
Vasopressin and sympathetic response 787 A.L. Garc a-Villalo n et al
was stimulation frequency, another factor was vasopressin
concentration and another presence or absence of the
antagonist. A probability value of less than 0.05 was
considered signicant.
Drugs used were: [Arg
8
]-vasopressin acetate; [deamino-
Cys
1
, D-Arg
8
]-vasopressin (desmopressin) acetate; the V
1
antagonist (b-Mercapto-b,b-cyclopenta-methylenepropionyl
1
,
O-Me-Tyr
2
,Arg
8
)-vasopressin [d(CH
2
)
5
Tyr(Me)AVP]; adeno-
sine 5'-triphosphate, disodium salt (ATP); (7)-arterenol,
bitartrate salt (noradrenaline); L-N
G
-nitro-arginine (L-
NOARG); nickel chloride hexahydrate (NiCl
2
); phentolamine
hydrochloride; prazosin hydrochloride; verapamil hydrochlor-
ide; and yohimbine hydrochloride; all from Sigma; pyridox-
alphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS
tetrasodium salt) from Tocris Cookson Ltd.; and 5-allyl-2-
amino-5,6,7,8-tetrahydro-4H-thiazolo-[4,5]-dazepin hydro-
chloride (BHT-920) was a gift from Europharma S.A.
Results
Response to electrical eld stimulation
Electrical stimulation (1 8 Hz) produced frequency-depen-
dent contraction of the vascular segments at 37 and 308C, but
at 308C the response was signicantly lower (P50.001) than at
378C, at every frequency of stimulation (Figure 1). For
example, the arterial contraction (8 Hz) at 308C was
0.59+0.06 g (n=32) and at 378C was 1.6+0.12 g (n=30:
P50.01).
Figure 2 Contraction of rabbit ear arteries at 37 and 308C to electrical eld stimulation (1 8 Hz, 0.2 ms pulse duration, 70 V, for
5 s) in the absence (control) and in the presence of: (a) phentolamine (1 mM); (b) prazosin (1 mM); and (c) yohimbine (1 mM). Points
are means+s.e.mean. *, **, Signicantly dierent from the control (*P50.05, **P50.01). n=number of animals.
Vasopressin and sympathetic response 788 A.L. Garc a-Villalo n et al
Eects of vasopressin on the response to electrical eld
stimulation
Control Vasopressin, at the concentrations used, did not
produce contraction, or in some cases produced a contraction
that markedly diminished after applying electrical stimulation.
If the contraction induced by vasopressin did not return to less
than 25% of the maximal response to electrical stimulation,
these data were discarded following the protocol used in a
previous study (Padilla et al., 1997).
In the presence of vasopressin (10 pM, 100 pM and 1 nM)
the contraction to electrical stimulation was greater than that
in the absence of the peptide (Figure 1a). At 378C, the
potentiating eect of vasopressin was signicant (P50.01)
only at 1 Hz for every vasopressin concentration, at 2 Hz for
vasopressin at 10 and 100 pM, and at 4 Hz only for vasopressin
at 10 pM. At 308C, vasopressin increased the contraction to
electrical stimulation in a concentration-dependent manner,
and this increase was signicant (P50.01) at every stimulation
frequency and vasopressin concentration. The increments
induced by vasopressin at 308C were signicantly higher
(P50.01) than at 378C for 4 and 8 Hz frequencies and 100 pM
and 1 nM vasopressin concentrations; for 1 Hz it was higher
(P50.05) at 378C for all vasopressin concentrations (Table 1).
Figure 3 Contraction of rabbit ear arteries at 37 and 308C to electrical eld stimulation (1 8 Hz, 0.2 ms pulse duration, 70 V, for
5 s) in the absence (control) and in the presence of: (a) PPADS (30 mM); and (b) PPADS (30 mM) plus phentolamine (1 mM). Points
are means+s.e.mean. *, **, Signicantly dierent from the control (*P50.05, **P50.01). n=number of animals.
Table 1 Vasopressin-induced increments in g, of the contraction of rabbit central ear arteries to electrical eld stimulation (1 8 Hz,
0.2 ms pulse duration, 70 V for 5 s) at 37 and 308C
378C (n=30) 308C (n=32)
Vasopressin 1Hz 2Hz 4Hz 8Hz 1Hz 2Hz 4Hz 8Hz
10 pM
100 pM
1 nM
0.14+0.03
0.31+0.04
0.33+0.08
0.16+0.03
0.22+0.05
0.14+0.12
0.11+0.04
0.04+0.05
70.01+0.13
0.05+0.04
0.01+0.06
70.06+0.1
0.02+0.01**
0.07+0.01**
0.18+0.03*
0.1+0.02
0.24+0.02
0.39+0.04*
0.15+0.02
0.32+0.03**
0.45+0.04**
0.16+0.02*
0.34+0.03**
0.45+0.03**
Values are means+s.e.mean. *,**, signicantly dierent from 378C (*P50.05; **P50.01). n=number of animals.
Vasopressin and sympathetic response 789 A.L. Garc a-Villalo n et al
Vasopressin receptor subtypes involved The antagonist of V
1
vasopressin receptors d(CH
2
)
5
Tyr(Me)AVP (100 nM) by itself
did not modify the contraction to electrical stimulation (data
not shown). In the presence of this antagonist, the potentiation
of the response to electrical stimulation produced by
vasopressin in control conditions was not present either at 37
or 308C (Figure 1b) (n=7).
The agonist of vasopressin V
2
receptors desmopressin did
not produce any contraction of rabbit ear artery and did not
modify the contraction to electrical stimulation at 37 and 308C
(Figure 1c) (n=7).
Adrenoceptor and purinoceptor blockade The contraction of
the ear artery to electrical stimulation was signicantly reduced
by application of phentolamine (1 mM, Figure 2a, P50.01,
n=6) or prazosin (1 mM, Figure 2b, P50.05, n=6), and was
signicantly increased (P50.01) by yohimbine (1 mM, Figure
2c, n=6), at both 37 and 308C. However, application of
PPADS (30 mM, n=7) slightly increased the response at 378C
(P50.01) and did not aect the response at 308C (Figure 3a).
Application of PPADS plus phentolamine markedly reduced
(P50.01, n=7) the contraction to electrical stimulation at
both temperatures (Figure 3b).
At 378C, the potentiation of the arterial contraction to
electrical stimulation by vasopressin was increased (P50.001)
by phentolamine (Figure 4a, n=6) or prazosin (Figure 4b,
n=6) but was not modied by yohimbine (Figure 4c, n=6). At
this temperature PPADS (P50.001, Figure 4d, n=7) or
PPADS plus phentolamine (P50.001, Figure 4e, n=7)
changed the eect of vasopressin on the contraction to
Figure 5 Contraction of rabbit ear arteries at 37 and 308C to
electrical eld stimulation (1 8 Hz, 0.2 ms pulse duration, 70 V, for
5 s) in arteries under control conditions and in arteries: (a) pretreated
with verapamil (10 mM); (b) pretreated with NiCl
2
(1 mM); (c) without
endothelium; and (d) pretreated with L-NOARG (100 mM). Points are
means+s.e.mean. *, **, Signicantly dierent from the control
(*P50.05, **P50.01). n=number of animals.
Figure 4 Contraction of rabbit ear arteries at 37 and 308C to
electrical eld stimulation (1 8 Hz, 0.2 ms pulse duration, 70 V, for
5 s) in the absence (control) and in the presence of vasopressin
(10 pM 1 nM) in arteries pretreated with: (a) phentolamine (1 mM);
(b) prazosin (1 mM); (c) yohimbine (1 mM); (d) PPADS (30 mM); and
(e) PPADS (30 mM) plus phentolamine (1 mM). Points are means
+s.e.mean. *, **, Signicantly dierent from the control (*P50.05,
**P50.01). n=number of animals.
Vasopressin and sympathetic response 790 A.L. Garc a-Villalo n et al
electrical stimulation, as during these two treatments
vasopressin reduced the response to electrical stimulation. At
308C, the potentiation produced by vasopressin was reduced
by phentolamine (P50.05, Figure 4a, n=6), yohimbine
(P50.05, Figure 4c, n=6) or PPADS (P50.001, Figure 4d,
n=7), was abolished by phentolamine plus PPADS (P50.001,
Figure 4e, n=7) and was not modied by prazosin (Figure 4c,
n=6).
Role Ca
2+
channels The specic blocker of L-type Ca
2+
channels verapamil (1 mM) did not modify the response of
vascular segments to electrical stimulation at 37 or 308C
(Figure 5a, n=6), whereas the non-specic Ca
2+
channel
blocker, NiCl
2
(1 mM) reduced the arterial response at 378C
(P50.01, n=6) but not at 308C (Figure 5b, n=6). At 308C, the
potentiating eect of vasopressin on the contraction of ear
arteries to electrical stimulation was reduced by verapamil
(P50.001, n=6) and abolished by NiCl
2
(P50.001, n=6). At
378C, in the arteries pretreated with verapamil (n=6) or NiCl
2
(n=6) vasopressin did not potentiate but reduced (P50.001)
the contraction to electrical stimulation (Figure 6a and b).
Nitric oxide synthesis inhibition and endothelium removal The
contraction of rabbit ear artery to electrical stimulation was
increased by endothelium removal at 308C (P50.01, n=6) but
not at 378C (Figure 5c, n=6), and was increased by
pretreatment of the arteries with L-NOARG (100 mM) at both
temperatures (P50.01, Figure 5d, n=6). However, the
potentiation by vasopressin was not signicantly modied by
endothelium removal (n=6) or L-NOARG (n=6) (Figure 6c
and d) compared to intact, non treated arteries, at both
temperatures.
Eect of vasopressin on the contraction to noradrenaline,
BHT-920 and ATP
A submaximal concentration of noradrenaline (1 mM) pro-
duced contraction of ear arteries, which was not signicantly
dierent (P40.05) at 378C (2.78+0.3 g, n=6) and 308C
(2.23+0.19 g, n=6), and this contraction was not modied in
the presence of vasopressin, either at 37 or 308C (Figure 7a,
n=6).
The selective a
2
-adrenoceptor agonist, BHT-920 (10 mM)
after treatment with prazosin (1 mM), produced a small
contraction at 378C (0.09+0.03 g, n=6) and no observable
response at 308C in control arteries (Figure 7b, n=6).
However, after pretreatment with both prazosin (1 mM) and
vasopressin, the response to BHT-920 was markedly increased
(P50.01, n=6) at 378C, and this adrenoceptor agonist
produced a clear contraction at 308C (n=6).
A submaximal concentration of ATP (2 mM) contracted
arterial segments at 378C (0.69+0.07 g, n=6) and 308C
(0.47+0.07 g, n=6). There was not statistically signicant
Figure 6 Contraction of rabbit ear arteries at 37 and 308C to
electrical eld stimulation (1 8 Hz, 0.2 ms pulse duration, 70 V, for
5 s) in arteries in the absence (control) and in the presence of
vasopressin (10 pM 1 nM): (a) pretreated with verapamil (10 mM); (b)
pretreated with NiCl
2
(1 mM); (c) without endothelium; and (d)
pretreated with L-NOARG (100 mM). Points are means+s.e.mean. *,
**, Signicantly dierent from the control (*P50.05, **P50.01).
n=number of animals.
Figure 7 Contraction of rabbit central ear arteries at 37 and 308C,
in the absence (control) and in the presence of vasopressin (10 pM
1 nM), in response to: (a) noradrenaline (1 mM); (b) BHT-920 (10 mM)
in the presence of prazosin (1 mM); and (c) ATP (2 mM). Values are
means+s.e.mean. *Signicantly dierent from its control (*P50.01).
n=number of animals.
Vasopressin and sympathetic response 791 A.L. Garc a-Villalo n et al
dierence in the response at these temperatures (P40.05). The
presence of vasopressin in the organ bath increased (P50.01)
the arterial contraction to ATP. The magnitude of this
increment was similar at both 37 and 308C (Figure 7c, n=6).
Discussion
The results of this study indicate that the contractile response
of rabbit ear arteries to sympathetic nerve stimulation was
reduced at 308C compared to 378C and that vasopressin
potentiated this response to a greater extent at 30 than at 378C,
for most of the frequencies of electrical stimulation used. These
results thus conrm a previous study from our laboratory
(Padilla et al., 1997). The main aim of this study was to analyse
the mechanisms underlying the potentiating eect of vaso-
pressin on sympathetic vasoconstriction.
Under control conditions, the characteristics of the response
of ear arteries to sympathetic stimulation at 37 and at 308C are
qualitatively similar to those reported in previous studies
(Garc a-Villalo n et al., 1997a,b). In those studies we suggested
that the reduction in the sympathetic response of these arteries
at 308C may be mainly due to a diminished participation of
postjunctional a
1
-adrenoceptors. Also, we suggested that a
purinoceptor component, in addition to the a
1
-adrenoceptor
component, may be present in the sympathetic response of
these arteries at 308C, and that this response is modulated by
endothelial nitric oxide release, to a greater extent at 30 than at
378C. The results of the present study and of one of those
studies (Garc a-Villalo n et al., 1997b) also suggest that there
may be inhibitory prejunctional a
2
-adrenoceptors and/or
purinoceptors in the nerve terminals of the rabbit ear artery
since the arterial response to electrical stimulation was
increased by purinoceptor inhibition at 378C, and by
yohimbine at both 30 and 378C. According to previous studies
from our and other laboratories, the concentrations of the
receptor antagonists or enzyme inhibitors used in the present
study are likely to be selective and/or eective: antagonists for
vasopressin V
1
receptors (100 nM, Garc a-Villalo n et al., 1996;
Mart nez et al., 1994), phentolamine (1 mM, Garc a-Villalo n et
al., 1997b), prazosin (1 mM, Garc a-Villalo n et al., 1997b;
Lefebvre & Smits, 1992), yohimbine (1 mM, DeMan et al.,
1994; Garc a-Villalo n et al., 1997b), PPADS (30 mM, Garc a-
Villalo n et al., 1997b), verapamil (10 mM, unpublished
observations from our laboratory), NiCl
2
(1 mM; Hirano et
al., 1989) and L-NOARG (100 mM; Padilla et al., 1998).
With regard to the mechanisms underlying the potentiation
of the sympathetic contraction of ear arteries by vasopressin,
our results suggest that this potentiation is mediated by
activation of vasopressin V
1
receptors, because this potentia-
tion was abolished by the vasopressin V
1
receptor antagonist
d(CH
2
)
5
Tyr(Me)AVP, whilst desmopressin, an agonist of
vasopressin V
2
receptors, did not aect the response of these
arteries to sympathetic stimulation. These results agree with
those found in mesenteric arteries of humans (Medina et al.,
1997) or rats (Noguera et al., 1997) in which vasopressin also
potentiated the contraction to adrenoceptor stimulation by
activation of vasopressin V
1
receptors.
Another aspect of the present work is the role of
adrenoceptors and purinoceptors in the potentiation of the
sympathetic contraction by vasopressin. In the rabbit ear
artery it has been shown previously that the constriction to
sympathetic stimulation may be mediated by release of both
noradrenaline and ATP from perivascular sympathetic nerve
terminals (Kennedy et al., 1986), and these transmitters then
act on a-adrenoceptors and purinoceptors, respectively. The
present study suggests that, at 378C, vasopressin did not
potentiate the response to a
1
-adrenoceptor activation. This
suggestion is supported by the observations that phentolamine
or prazosin increased instead of reduced the potentiating eect
of vasopressin on the response to nerve stimulation, and that
vasopressin did not modify the arterial contraction to
exogenous noradrenaline (this amine contracts rabbit ear
arteries by activating mainly a
1
-adrenoceptors, Garc a-Villalo n
et al., 1992). On the other hand, at 378C, the potentiating eect
of vasopressin on nerve stimulation was abolished by PPADS,
and at the same time, vasopressin increased the contraction to
exogenous ATP. This suggests that vasopressin, at this
temperature, potentiates the contraction to sympathetic
stimulation mainly by facilitating the response to purinoceptor
activation. This might explain why the potentiation by
vasopressin at 378C is greater at the lower stimulation
frequencies than at the higher ones (Table 1), as the purinergic
component of the sympathetic response may be more apparent
at lower frequencies of stimulation (Kennedy et al., 1986). The
involvement of a
2
-adrenoceptors in the potentiating eect of
vasopressin at 378C is unclear, since this eect of vasopressin
was not modied by yohimbine, but vasopressin did potentiate
the contraction to BHT-920.
At 308C, the mechanisms underlying the vasopressin eect
on sympathetic stimulation may dier from those at 378C. Our
data with PPADS and ATP suggest that at 308C, vasopressin
potentiated the purinoceptor component of the sympathetic
contraction, and that this potentiation for high stimulation
frequencies (4 and 8 Hz) was more marked at 30 than at 378C.
Moreover, our results at 308C suggest that, in addition to
potentiating the purinoceptor component, vasopressin may
also potentiate the response to a
2
-adrenoceptor activation.
This is suggested by the observation at 308C that the
potentiation of the sympathetic response by vasopressin was
reduced by phentolamine and yohimbine but not by prazosin,
and that the potentiating eect of vasopressin still present after
PPADS treatment was abolished by phentolamine. This is in
line with the results of Guc et al. (1992) who observed, in
pithed rats, that vasopressin increased the pressor eects of
noradrenaline, but not that due to selective stimulation of a
1
-
adrenoceptors.
Regarding the role of Ca
2+
channels, our results at 37 and
308C suggest that the potentiation of the vasoconstriction to
sympathetic stimulation by vasopressin is mediated by entry of
extracellular Ca
2+
, at least in part through Ca
2+
channels of
the L-type, because the vasopressin eect was reduced by the
Ca
2+
channels blockers verapamil and NiCl
2
, at both
temperatures. Results reported in the literature in this respect
show that the potentiating eect of vasopressin on the
contraction to adrenoceptor stimulation was mediated by L-
type Ca
2+
channels in rat (Medina et al., 1997) but not in
human (Noguera et al., 1997) mesenteric arteries, suggesting
that species dierences may be involved. Our results also
suggest that T-type Ca
2+
channels might be also involved in
the potentiating eect of vasopressin at 308C, since at this
temperature NiCl
2
was more eective than verapamil in
inhibiting the potentiation by vasopressin. To clarify this
question, however, antagonists more specic for this subtype
of Ca
2+
channels should be used.
In relation to the role of the vascular endothelium and nitric
oxide, we have found in a previous study (Garc a-Villalo n et
al., 1996) that the modulatory role of nitric oxide in the eect
of vasopressin on rabbit arteries varies widely between
vascular beds, and that this modulatory role of nitric oxide
was particularly small in ear arteries. In line with this, the
present results at 37 and 308C suggest that the role of the
Vasopressin and sympathetic response 792 A.L. Garc a-Villalo n et al
endothelium and nitric oxide may be of relatively little
importance for the potentiating eect of vasopressin on the
sympathetic contraction of ear arteries, as this potentiation
was not modied by endothelium removal nor by nitric oxide
synthase inhibition, at both temperatures. However, nitric
oxide may modulate, at both temperatures, the response of ear
arteries to sympathetic stimulation, since nitric oxide synthase
inhibition in the absence of vasopressin did increase the
response to electrical stimulation. Thus, it is suggested that, at
37 and 308C, nitric oxide release may be stimulated by
sympathetic stimulation to a similar extent in the absence and
in the presence of vasopressin.
Therefore, it may be suggested that vasopressin potentiates
the contraction of cutaneous (ear) arteries to sympathetic
stimulation, to a greater extent at 30 than at 378C. At both
temperatures, this potentiating eect of vasopressin may be
mediated by activation of vasopressin V
1
receptors and Ca
2+
channels, and it may be independent of endothelial nitric
oxide. The increased potentiating eect of vasopressin at 308C
may not be due to cooling-induced changes of vasopressin
receptor subtype, nor to changes of Ca
2+
channels or
endothelial nitric oxide eects. It may be hypothesized,
instead, that cooling (308C) changes the facilitating action of
vasopressin on the receptors that mediate the sympathetic
response of cutaneous arteries, so at normal temperature
(378C) vasopressin would potentiate mainly purinoceptor
eects whereas during cooling (308C) it would potentiate both
purinoceptor and a
2
-adrenoceptor eects.
The authors are grateful to Mrs M.E. Mart nez and H. Ferna ndez-
Lomana for technical assistance. This work was supported, in part,
by FIS ((6/0474), DGICYT (PM 95/0032), and CAM (AE 263/95).
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(Received May 20, 1998
Revised October 1, 1998
Accepted November 6, 1998)
Vasopressin and sympathetic response 793 A.L. Garc a-Villalo n et al