Neurochemical Modulation of Central
Cardiovascular Control: The Integrative
Role of Galanin
Zaida Dıaz-Cabiale, Concepcion Parrado, Manuel Narvaez, Carmelo Millon,
Araceli Puigcerver, Kjell Fuxe, and Jose Angel Narvaez
Abstract Galanin (GAL) is a peptide involved in multiple functions, including
central cardiovascular control. In this review, the role of GAL and its fragments in
the modulation of cardiovascular neuronal networks in the nucleus of the solitary
tract is presented, including its interaction with the classical neurotransmitters and
other neuropeptides involved in cardiovascular responses in this nucleus. First, we
describe the cardiovascular responses of GAL and the pathway involved in these
responses. Then we summarize findings obtained in our laboratory on how GAL,
through its receptors, interacts with two other neuropeptides – Neuropeptide Y and
Angiotensin II and their receptors – as they have particularly conspicuous cardio-
vascular effects. All these results strengthen the role of GAL in central cardiovas-
cular control and indicate the existence of interactions among GAL receptor
subtypes and a2-adrenergic receptors, AT1, and Y1 receptor subtypes. These
interactions are crucial for understanding the integrative mechanisms responsible
for the organization of the cardiovascular responses from the NTS.
Keywords Galanin fragments � NTS � Cardiovascular control � Angiotensin II �NPY
Z. Dıaz-Cabiale, M. Narvaez, C. Millon, and J.A. Narvaez (*)
Department of Physiology, University of Malaga, Malaga, Spain
e-mail: [email protected]
C. Parrado
Department of Histology, University of Malaga, Malaga, Spain
A. Puigcerver
Department of Psychobiology, University of Malaga, Malaga, Spain
K. Fuxe
Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden
T. Hokfelt (ed.), Galanin, Experientia Supplementum 102,
DOI 10.1007/978-3-0346-0228-0_9, # Springer Basel AG 2010
113
Introduction
Central control of cardiovascular function has been studied for a number of
decades. Of particular interest are the homeostatic control mechanisms such as
the baroreceptor-heart rate reflex and the chemoreceptor reflex. Blood pressure
homeostasis is maintained with the participation of several brain regions and a
variety of neurotransmitters and neuropeptides; these can possess either the same or
opposing functions when released from the central nervous system neurons.
The nucleus of the solitary tract (NTS) plays a critical role in integrating
peripherally initiated sensory information such as the status of blood pressure,
heart rate, and respiratory function. In fact, projections from receptors in the carotid
sinus, the aortic arch and cardiopulmonary sites reach the NTS, within which the
first synapse of the baroreflex loop is located [1–3].
Short neuronal loops which link the baroreceptor afferents and efferents also
exist. Thus, the NTS neurons innervate the parasympathetic motor centers, namely
the dorsomedial motor nucleus of the vagus and the nucleus ambiguus, and are
reciprocally interconnected with therostral ventrolateral medulla and the caudal
ventrolateral medulla [1–3].
Other areas involved in central cardiovascular control are the medullary raphe,
the A5 noradrenergic neurons in the pons, and the parvicellular paraventricular
nucleus of the hypothalamus where descending monosynaptic inputs to spinal
sympathetic preganglionic vasomotor neurons partly arise [2]. The NTS innervates
these areas either directly or indirectly and these connections are essential for
integrated cardiovascular and behavioral responses.
Many endogenous neurotransmitters such as catecholamines, serotonin, gamma
aminobutyric acid and glutamate, among others, have the ability to modulate blood
pressure and heart rate at the level of the NTS [3, 4].
Furthermore, most of the known mammalian peptidergic neuronal systems have
been found to exist in the NTS. They have been thought to be involved in the
transmission processes of this nucleus, with probable participation in its multiple
regulatory mechanisms. Detailed cardiovascular studies have been performed with
some of these peptides, including Galanin (GAL), in the NTS [4].
In this review, the role of GAL in the modulation of cardiovascular neuronal
networks in NTS is presented, including its interaction with the classical neuro-
transmitters and other neuropeptides involved in cardiovascular responses in this
nucleus. First, we describe the cardiovascular responses of GAL and the pathway
involved in these responses. Then we will summarize findings obtained in our
laboratory on how GAL, through their receptors, interacts with other neuropeptides.
Two of them, Neuropeptide Y (NPY) and Angiotensin II (Ang II) and their
receptors have been selected as they are particularly conspicuous with respect to
their cardiovascular effects. All these results strengthen the role of GAL in central
cardiovascular control and provide a better understanding of the transmission
mechanisms responsible for the organization of the cardiovascular responses from
the NTS.
114 Z. Dıaz-Cabiale et al.
Role of Galanin in Central Cardiovascular Regulation
A possible role of GAL in cardiovascular regulation has been studied as both GAL-
like immunoreactivity and the GAL receptors show a wide distribution in the
NTS [5].
Intracisternal injections of GAL elicit a transient vasopressor response followed
by a rapid decrease in mean arterial pressure (MAP) [6, 7]. This early increase of
MAP appeared 5 min after the injections but from the tenth minute a progressive
decrease in MAP was observed. The decrease in MAP was accompanied by
tachycardia, but this effect cannot be described as a reflex because tachycardia
appeared even at doses that failed to elicit changes in blood pressure [6–8].
In recent experiments to determine the site of action of GAL, we analyzed the
effects of microinjections of GAL into the NTS in anaesthetized rats. We observed
that GAL, at the dose of 10 pmol, induced an increase in MAP (p < 0.01). This
response was maintained during the 30 min recording period and was observed at a
dose of 20 pmol also (p < 0.05) (Fig. 1).
These results confirm a role for GAL in cardiovascular control in the NTS.
However, as a vasodepressor action was observed after intracerebroventricular
injections, other nuclei in the brainstem should also be involved.
Three cloned receptor subtypes for Galanin - GALR1, GALR2 and GALR3 are
expressed in the NTS [9–11], and both GALR1 and GALR3 signal via Gai-proteindecrease cyclic AMP levels by inhibiting adenylate cyclase [12]. In the NTS,
GALR1 inhibits N- and P/Q-types of voltage-dependent Ca2+ channels [13]. On the
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Galanin (1-29) (pmol)5 10 20
*
**
Fig. 1 Effects of microinjections into the NTS of different doses of Galanin and aCSF on MAP
pressure over a 30 min recording period. Means � S.E.M. are shown as percentages of changes
from the respective basal values. n ¼ 6–8 rats per group. The basal values were: Galanin 1 pmol
group 77 � 2 mmHg; Galanin 2.5 pmol group 78 � 1 mmHg; Galanin 5 pmol group 80 � 3
mmHg; Galanin 10 pmol group 73 � 2 mmHg; Galanin 20 pmol group 85 � 6 mmHg and aCSF
group 80 � 6 mmHg. *p< 0.05, **p< 0.01 vs. the control group according to one-way ANOVA
followed by Fisher post test
Neurochemical Modulation of Central Cardiovascular Control 115
other hand, the main pathway downstream from GAL-R2 involves coupling to Gaq/11-protein which activates phospholipase C resulting in inositol triphosphate accu-
mulation and subsequent increase of intracellular Ca2+ [14]. This would presumably
stimulate neuronal activity and neurotransmitter release. Although we cannot exclude
the participation of any of them in this response, it could be hypothesized that the
GALR2 is the main receptor involved in the vasopressor response induced by GAL in
the NTS.
With regard to heart rate (HR), no effects were observed after the microinjection
of GAL in the NTS. As a tachycardic response was observed after the intracisternal
microinjections of GAL, it may be proposed that it is probably the dmnX and not
the NTS the main nucleus involved in the HR response.
To elucidate the efferent pathways responsible for GAL-induced tachycardic
response, the effect of intracisternal GAL was examined in rats pretreated with
atropine or propranolol [15]. As seen in Fig. 2, while pretreatment with propranolol
did not modify changes in MAP or HR elicited by GAL, pretreatment with atropine
induced a significant vasopressor and tachycardic response that was maintained
during the whole recording period (Fig. 2). These results confirm the involvement
of parasympathetic pathways in mediating the MAP, and HR response elicited by
intracisternal GAL [15]. Previous work has also suggested that GAL actions might
be mediated through the parasympathetic pathways, as intravenous GAL mimicked
the attenuation of cardiac vagal activity following a period of sympathetic nerve
stimulation [16, 17].
Fig. 2 Representative tracings of the effect of GAL (3 nmol/rat) in nontreated rats (a) and in rats
given atropine (125 mg/kg); (b), and propranolol (1 mg/kg); (c). The increases in HR elicited by
intracisternal GAL is modified after pretreatment with atropine but not after pretreatment with
propranolol. Figure reproduced from [15] with permission
116 Z. Dıaz-Cabiale et al.
However, GAL activity in sympathetic pathways cannot be excluded as it has
recently been shown that GAL reduces sympathetic vasomotor tone by acting on
the rostral ventrolateral medulla [18].
GAL is also involved in the cardiovascular control of other brain areas. It has
been shown that hypothalamic paraventricular nucleus galaninergic projections to
NTS suppress baroreceptor reflex [19]. Also, galaninergic projections to the NTS
participate in the suppression of the baroreceptor reflex response by the locus
coeruleus [20].
GAL Fragments
In addition to GAL, GAL fragments have also been shown to be active in central
cardiovascular control. It has been demonstrated that intracisternal N-terminal
fragments of GAL such as (1–15) or GAL (1–16) produce a vasopressor response
significantly different from the response induced by GAL, whereas C-terminal
fragments are inactive (Fig. 3) [8, 21]. Both N-terminal GAL fragments induced
a tachycardic response which was similar in intensity to that observed with GAL
[21]. The nucleus involved in the changes in MAP and HR induced by GAL (1–15)
seems to be the NTS as microinjections of GAL (1–15) in this nucleus reproduce a
significant vasopressor (Fig. 4) and tachycardic response. Although the three cloned
30
15
Galanin(1-15) Galanin(1-29)
Galanin(10-29)Galanin(1-16)
CONTROL
p<0.05
p<0.01
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Fig. 3 Effects of intracisternal injections of porcine galanin (1–29) (▪), the N-terminal galanin
fragment (1–15) (l), the N-terminal galanin fragment (1–16) (m), or the C-terminal galanin
fragment (10–29) (□) (3 nmol/rat in all cases) on mean arterial blood pressure over a 60 min
recording period. Means � S.E.M. are shown as percentages of changes from the respective basal
values. n ¼ 6–8 rats per group. The basal values were: control group 86 � 6 mmHg; galanin-
(1–15) group 85 � 3 mmHg; galanin-(1–16) group 90 � 10 mmHg; galanin-(1–29) group
90 � 3 mmHg; galanin-(10–29) group 88 � 10 mmHg. p< 0.01 and p< 0.05 are the significance
level of the gal-(1–15) and gal-(1–16) groups vs. control group (Fisher post hoc-test), when
considering the entire observation period. Figure reproduced from [21] with permission
Neurochemical Modulation of Central Cardiovascular Control 117
receptors for GAL have a higher affinity for GAL than for GAL fragments, specific125I-Galanin (1–15) binding sites have been described in the central nervous system
[22]. In the brainstem, a cluster of these specific sites appears within the NTS, but
not in the ventrolateral medulla [22] suggesting that this is a primary site for GAL
(1–15) binding. This is in agreement with the results obtained with microinjections
of GAL (1–15) in the NTS inducing an increase in MAP and HR.
Thus, it seems that both GAL and GAL (1–15) molecules have specific roles in
cardiovascular regulation [23]. Also, threshold doses of the N-terminal fragment
GAL (1–15) were able to antagonize the cardiovascular effects of whole molecule
GAL [8]. In fact, pretreatment with propranolol, a b-adrenoreceptor antagonist,blocks the cardiovascular effects elicited by the N-terminal fragment GAL (1–15),
but not the effects elicited by GAL, while pretreatment with the cholinergic
antagonist, atropine, modifies the changes induced by GAL but not by GAL
(1–15) [15, 23]. It has been demonstrated that GAL and GAL (1–15) stimulate
the expression of c-fos with different temporal and spatial profiles, especially in the
NTS and in the catecholaminergic area of the ventrolateral medulla [24].These
differences suggest the possible existence of novel GAL receptor subtypes that
preferentially bind to the N terminal GAL fragment. In line with this view, the
specific GAL receptor antagonist, M40, was able to block cardiovascular responses
elicited by the N-terminal fragment GAL (1–15), but not those elicited by the whole
GAL molecule [25]. These results support the hypothesis of the existence of one
receptor with a higher affinity for the N-terminal fragment than for GAL. This
receptor remains to be cloned [23]. However, we have proposed that this GAL
25
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Galanin (1-15) (pmol)1 2.5 5 10
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Fig. 4 Effects of microinjections into the NTS of different doses of Galanin (1–15) and aCSF on
MAP pressure over a 30 min recording period. Means � S.E.M. are shown as percentages of
changes from the respective basal values. n ¼ 6–8 rats per group. The basal values were: GAL
(1–15) 0.5 pmol group 84 � 5 mmHg; GAL (1–15) 1 pmol group 79 � 3 mmHg; GAL (1–15)
2.5 pmol group 77 � 1 mmHg; GAL (1–15) 5 pmol group 81 � 4 mmHg; GAL (1–15) 10 pmol
group 75 � 3 mmHg; and aCSF group 80 � 6 mmHg. ***p < 0.001 vs. the control group
according to one-way ANOVA followed by Fisher post test
118 Z. Dıaz-Cabiale et al.
receptor subtype is the result of the formation of a GALR1–GALR2 heterodimer
which has developed a preferential affinity for the N terminal GAL fragment [26].
Galanin/Classical Neurotransmitter Interactions in Central
Cardiovascular Regulation
The interaction of neuropeptides with several neurotransmitters such as serotonin
and noradrenaline has been proposed as an important mechanism involved in the
integration of the cardiovascular responses in the NTS [4, 27]. The possible inter-
actions of GAL with serotonin 5-HT1A receptors and noradrenaline a2-adrenore-ceptors have therefore been analyzed at the cardiovascular level.
GalaninR/5-HT1A Interactions
It is likely that all serotonin receptors, except the 5-HT6 type, are involved in
cardiovascular regulation [28]. The central 5-HT1A receptor plays a physiological
role in the regulation of cardiovascular reflexes, controlling changes in parasympa-
thetic drive to the heart [28]. These reflexes also affect activity in the sympathetic
nervous system, which itself can be inhibited by central 5-HT1A receptors resulting in
decreased blood pressure [28]. After intracisternal co-injections of GAL and 5-HT1A
receptor selective agonist 8-OH-DPAT, a significant vasodepressor response was
observed [7] indicating that GALR and 5-HT1A receptors interact in the control of
central cardiovascular mechanisms. This interaction is bidirectional as the presence of
8-OH-DPAT increases the binding of 125I Galanin, inter alia, in the NTS [7]. Also,
GAL (1–15) enhances the hypotension elicited by 8-OH-DPAT injected intracister-
nally but antagonizes its bradycardic response, leading to tachycardia [8].
These interactions between GALR and 5-HT1A have been explained based on
the existence of GALR/5-HT1A heteromers in which direct allosteric receptor–
receptor interactions occur [26]. In cardiovascular networks a facilitatory 5-HT1A/
GALR interaction enhancing GAL receptor affinity and efficacy has been observed.
Thus, GAL and the 5-HT1A receptor agonist 8-OH-DPAT, when co-injected intra-
cisternally, act synergistically to produce vasodepressor responses which can be
explained by the existence of such GAL/5-HT1A heteromers in the central cardio-
vascular region. Thus, GALR/5-HT1A receptor interactions as heteromers represent
a novel integrative mechanism in 5-HT neurotransmission [26].
GalaninR/a2-Adrenoreceptor Interactions
Anatomical evidence indicates that two-thirds of the medial NTS is innervated
by catecholamine axonal terminals that contain mainly noradrenaline [29, 30], and
Neurochemical Modulation of Central Cardiovascular Control 119
some possibly adrenaline [31, 32]. Central administration of adrenaline and nor-
adrenaline produced vasodepressor and bradycardic responses, and these effects
seem to be meditated by a2-adrenergic receptors [33–35]. In the NTS, these
receptors are, inter alia, located in the cathecholaminergic cell groups A2/C2
[36]. It is known that the densities of a2-adrenergic receptors exceed those of
a1-adrenergic and b-adrenergic receptors in the NTS. The use of specific agonists
or antagonists has also demonstrated a pivotal role for a2-adrenergic receptors on
cardiovascular regulation. Thus activation of the a2-adrenergic receptor reduces
sympathetic tone [37] and the central administration of a2-adrenergic antagonists
elicits hypertension and tachycardia. As GAL in the NTS is, in part, co-stored in
catecholaminergic cells and co-released with catecholamines [38] the effects of the
interactions between GAL and a2-adrenergic receptors on cardiovascular responseshave been analyzed.
Intracisternal co-injections of threshold doses of GAL with a hypotensive dose of
the a2-adrenergic receptor agonist clonidine were found to induce a rapid vasopres-sor and tachycardic response [39]. No effects were observed with GAL (1–15) [39].
GAL was able to modify the binding characteristics of a2-adrenergic receptor in theNTS as GAL decreases the affinity of the a2-adrenergic receptor agonist binding
sites, and this results in a significant increase in the number of agonist binding sites
[39]. Evidence for the involvement of GALR was obtained through the demonstra-
tion that the specific GAL receptor antagonist M35 blocked the observed increase in
the a2-adrenergic receptor agonist binding sites induced by GAL [39].
Thus, an antagonistic GALR/a2-adrenergic receptor interaction existing in a
receptor heteromer could play a role in central cardiovascular control, with GAL
reducing the affinity and increasing the number of a2-adrenergic receptor agonist
binding sites in the NTS [26, 39]. The integrated signal of the GALR/a2-adrenergicreceptor is linked to a blockade by GAL of the a2-adrenoreceptors agonist inducedvasodepressor responses. A facilitatory reciprocal interaction was also observed, in
which the a2-adrenergic receptor agonist could increase GALR binding; this may
in turn enhance the tachycardic responses to intracisternal GAL. The postulated
GALR/a2-adrenergic receptor heteromer with allosteric reciprocal receptor–
receptor interactions may represent a general mechanism in the CNS since similar
antagonistic GALR/a2-adrenergic receptor interactions have also been observed
in the tel- and diencephalon which also were blocked by GAL receptor antago-
nists [40].
Galanin/Neuropeptide Interactions in Central
Cardiovascular Regulation
Neuropeptides are known to produce cardiovascular effects. Therefore, the possible
existence of interactions between GAL receptors and other neuropeptide receptors
with well-known cardiovascular effects, like Angiotensin II AT1 receptors and
NPY Y1 receptors, have also been analyzed.
120 Z. Dıaz-Cabiale et al.
GalaninR/Angiotensin II AT1 Receptor Interactions
Angiotensin II (Ang II) is a neuropeptide known to be involved in central cardio-
vascular control [41]. Ang II can influence arterial pressure at any one of a number
of sites in the brain. Microinjection of Ang II into the lateral or third ventricle,
hypothalamic PVN, rostral ventrolateral medulla, and the area postrema increases
arterial pressure [41, 42]. In the NTS, Ang II microinjection produces vasopressor
and tachycardic responses at nanomolar doses [43], but elicits vasodepressor and
bradycardic responses at picomolar doses [44]. Furthermore, microinjections of
Ang II into the NTS reduce baroreceptor reflex sensitivity [45], and endogenous
Ang II exerts a tonic inhibitory modulation of the baroreceptor reflex mediated by
AT1 receptors but not AT2 receptors [46].
Ang II is known to interact with other neuropeptides involved in cardiovascular
functions such as NPY [47] and therefore the interaction between GAL and Ang II
was analyzed. We observed that the transient vasopressor response produced by
GAL alone disappeared in the presence of Ang II (Fig. 5) suggesting a functional
interaction between these peptides. The Ang II receptor involved seems to be the
AT1 as the presence of the specific AT1 receptor antagonist DuP753 reversed the
response induced by Ang II (Fig. 5) [48].
The interactions of GAL with Ang II have also been observed in other areas
[49]. Intracerebroventricular injections of GAL inhibited Ang II-induced drinking
behavior and cardiovascular regulation in conscious rats [49]. One of the areas
considered to be involved in this response is the subfornical organ as it has been
demonstrated by electrophysiological studies that GAL inhibits the activity of Ang-
sensitive neurons in this area [50].
On the contrary, intracisternal co-injections of threshold doses of Ang II with
GAL(1–15), induce a significant vasopressor response that is maintained during
the whole recording period, without any significant effect on heart rate [48]. This
response was blocked by the AT1 specific antagonist DuP753 (Fig. 6) confirming
the involvement of the AT1 receptor subtype in the interaction [48].
It is possible that these interactions take place at the membrane level and a model
can be proposed for these interactions, based on the existence of receptor hetero-
dimers and higher-order heteromers (receptor mosaics) [26, 27, 48]. It has already
been shown that heterodimerization occurs between neuropeptide receptors. Thus,
the AT1 receptor can exist as a heterodimer with Bradykinin2 receptors (B2) [51];
this may explain the previously reported AT1/Bradykinin2 receptor interactions
[52, 53]. Also some GAL receptors (GALR1) are present as dimers [54].
Similar heterodimerization and/or receptor mosaic formations can explain
the results obtained for the interaction between AT1 and GAL receptor subtypes
[48]. In this case, AT1 activation can induce conformational changes in the GAL
receptor, decreasing its signaling through the whole GAL molecule but at the
same time increasing the signaling over the putative GAL-like receptor which
recognizes N-terminal GAL fragments (postulated to be a GALR1–GALR2
heterodimer) [26].
Neurochemical Modulation of Central Cardiovascular Control 121
The findings highlight the effect of GAL and GAL (1–15) on cardiovascular
control and point towards the existence of a modulatory effect of GAL and GAL
(1–15) on cardiovascular responses to Ang II based on the existence of allosteric
receptor–receptor interactions as receptor heterodimers and/or higher-order hetero-
mers of AT1 and GAL receptor subtypes.
GalaninR/Neuropeptide Y R Subtype Interactions
NPY is a peptide of 36 amino acid residues and was isolated from the porcine
intestine [55]. Its presence in the mammalian central nervous system (CNS) and the
existence of heterogeneously distributed different receptor subtypes have been
Fig. 5 Representative tracings of the effect of GAL (3 nmol/rat) (a), Ang II (3 nmol/rat) þ GAL
(3 nmol/rat) (b), and Ang II (3 nmol/rat) þ GAL (3 nmol/rat) þ DuP 753 (0.5 mg/rat) (c). Theincrease of MAP elicited by GAL disappears in the presence of Ang II, but is observed again in
presence of DuP753. Figure reproduced from [48] with permission
122 Z. Dıaz-Cabiale et al.
demonstrated [56]. In the NTS, the presence of Y1 and Y2 receptor subtypes has
been demonstrated. Previous studies demonstrated that intracisternal injections of
NPY induce a pronounced and long-lasting, dose-dependent decrease in blood
pressure and heart rate, as compared with the hypotension elicited by a2-adrenor-eceptor agonists or by adrenaline [57, 58]. Later on, it was shown that this
hypotensive effect was mimicked by injections of the specific Y1 agonist Leu31–
Pro34–NPY in the NTS [58] suggesting that the hypotensive effect of NPY was
mediated by the Y1 receptor subtype.
The Y2 receptor agonists induce different cardiovascular responses when injected
in the NTS. After the injection of the specific Y2 agonist NPY (13–36), a biphasic
response was observed, with an increase in blood pressure in the femtomolar range
Fig. 6 Representative tracings of the effect of GAL(1–15) (a); Ang II (3 nmol/rat) þ GAL(1–15)
(0.1 nmol/rat) (b), and Ang II (3 nmol/rat) þ GAL(1–15) (0.1 nmol/rat) þ DuP753 (0.5 mg/rat)(c). The increase of MAP elicited by Ang II + GAL(1–15) is counteracted by DuP753. Figure
reproduced from [48] with permission
Neurochemical Modulation of Central Cardiovascular Control 123
followed by a decrease at higher doses (picomolar range) [59, 60]. Moreover, the
stimulation of Y2 receptors counteracted the cardiovascular actions mediated by Y1
receptors [57].
Furthermore, the specific agonist NPY Y2 (13–36) was able to counteract the
hypotensive response induced by L-glutamate. This fact suggests that this NPY
receptor subtype could play a role in modulating the baroreceptor reflex [60].
To analyze the potential role of GAL in the modulation of these circuits in the
NTS, the effects of GAL on NPY Y1 and Y2 receptor activation were evaluated by
quantitative receptor autoradiography and cardiovascular analysis [61].
At the cardiovascular level, intracisternal co-injection of threshold doses of NPY
and GAL induced a significant vasopressor response, reaching a maximum of 40%
increase (Fig. 7) [61]. This effect was reproduced with the co-injection of a Y1
agonist and GAL after which an increase in MAP of the same magnitude was
observed, as with threshold doses of NPY and GAL. The co-injection of threshold
doses of GAL and a Y2 agonist only led to a weak increase in MAP. These results
suggest a specific interaction of GAL receptors with the NPY Y1 receptor subtype.
(Fig. 7) [61]. With respect to central HR regulation, the functional interaction of
GAL and NPY receptor was found to be specific for the NPY Y1 receptor subtype
(Fig. 7).
These interactions take place at the receptor level in the NTS as the results of
quantitative autoradiography showed that GAL decreased NPY-Y1 agonist binding
in the NTS [61]. This effect, mediated by GAL, seemed to be specific for the NPY
Y1 receptor subtype, as NPY Y2 agonist binding was not modified in the presence of
this effective concentration of GAL [61].
This interaction may again be based on the formation of heterodimers, in this
case, composed of GAL R and NPY Y1 receptors [26, 27], wherein GAL-induced
conformational change in the GAL receptor can pass over the receptor interface to
cause a conformational change in the NPY Y1 receptor, which in turn leads to
reduced Y1 recognition and G-protein coupling and thus to reduced NPY Y1
receptor signaling. Evidence for the involvement of GAL receptors was again
obtained through the demonstration that the antagonist M35 blocked the decrease
in NPY Y1 agonist binding induced by GAL [61].
At the cellular level, evidence for the interactions between NPY Y1 agonist and
GAL was obtained when coinjection of the two led to a decrease in c-Fos IR in the
medial NTS (Fig. 8) [61]. This result may reflect a reduction in the activity of the
vasodepressor systems due to the antagonistic GAL receptor–NPY Y1 receptor
interaction leading to reduction in Y1 receptor signaling.
Conclusions
The findings presented in this review demonstrate the role of GAL in central
cardiovascular control, indicate the existence of interactions between GAL receptor
subtypes and a2-adrenergic receptor, Ang II AT1 and NPY receptor subtypes, and
124 Z. Dıaz-Cabiale et al.
a
b
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60
20
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NPY(1-36) 7.5pmol
GAL (1-29) 3nmol
NPY(13-36) 7.5pmol
NPY(1-36) 7.5pmol+GAL(1-29) 3nmol
NPY(13-36) 7.5 pmol+GAL(1-29) 3nmol
Leu31,Pro34 NPY 7.5pmolLeu31,Pro34 NPY 7.5pmol+GAL(1-29) 3nmol
30
20
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Fig. 7 Effects of intracisternal injections of NPY(1–36) (7.5 pmol/rat); Leu31, Pro34 NPY
(7.5 pmol/rat); NPY (13–36) (7.5 pmol/rat); GAL(1–29) (3 nmol/rat); NPY(1–36) (7.5 pmol/
rat) þ GAL(1–29) (3 nmol/rat); Leu31, Pro34 NPY(7.5 pmol/rat) þ GAL(1–29) (3 nmol/rat);
NPY (13–36) (7.5 pmol/rat) þ GAL(1–29) (3 nmol/rat) on mean arterial blood pressure (MAP)
(a) and Heart rate (HR) (b). The peak effect is shown as percent changes from respective basal
values. Means � S.E.M. are given. n ¼ 6–8 rats per group. ***p< 0.001, **p< 0.01; *p< 0.05
according to one-way ANOVA followed by Fisher post test. Figure reproduced from [61] with
permission
Neurochemical Modulation of Central Cardiovascular Control 125
document the functional relevance of receptor interactions in central cardiovascular
control in the NTS.
In most of the interactions studied, with GAL there is a modulation by the agonist
activated GAL receptor of receptor binding of other receptors. Both antagonistic and
facilitatory receptor–receptor interactions have been demonstrated and it is pro-
posed that they take place in the corresponding heterodimers and/or higher order
heteromers of unknown stoichiometry and topology (receptor mosaics) [27]. This
hypothesis is presently being tested. The existence of dimers or heterodimers of
GALR, Y1 and AT1 receptors has been demonstrated. GAL receptor 1 (GalR1) [54]
can exist as a dimer and the Y1 receptor has been shown to exist as homodimers and/
or heterodimers with other members of the NPY receptor family [62]. Finally the
Ang II receptor AT1 has been shown to form a heterodimer with B2 receptors [51].
It is of substantial interest that both Y1 and GAL receptors antagonize
a2-adrenergic receptor recognition and signaling [39, 57] . This may take place at
the postulated Y1-a2-adrenergic receptor and GALR-a2-adrenergic receptor hetero-dimers respectively, and/or postulated Y1-a2-adrenergic receptor–GALR receptor
a
b
AP
NTS
GAL
Y1 AGONIST
GAL+Y1 AGONIST
c
d
Fig. 8 Photomicrographs
represent equivalent sections
through the nucleus of the
solitary tract (NTS) showing
c-FOS IR nuclei after i.c.v.
injection of GAL (A, B), Y1
agonist Leu31, Pro34 NPY
(c) and the coinjection of
GAL and Y1 agonist Leu31,
Pro34 NPY (d). The
coinjection of GAL and the
Y1 agonist reduced the c-Fos
IR induced by either of the
two peptides. AP area
postrema. The dashed box in(a) is the magnified area
represented in (b). Scale bar,100 mm. Figure reproduced
from [61] with permission
126 Z. Dıaz-Cabiale et al.
mosaics. It is probable that under circumstances of large catecholamine release in
the NTS, overstimulation of a2-adrenergic receptors occurs, leading to marked
reduction in blood pressure and bradycardia. Under the same conditions, increased
amounts of NPY and/or GAL will be co-released with CA to counteract and balance
the strong a2-adrenergic receptor activation and exaggerated vasodepressor res-
ponses by means of antagonistic GALR/a2R and NPY Y1/a2R interactions [27].
The fact that both neuropeptides, via their respective receptors, have almost the
same antagonistic effect on a2-adrenergic receptor stimulation suggests the possible
existence of redundant receptor circuits to maintain a proper adjustment of cardio-
vascular responses of high safety as to a2-adrenergic receptor induced cardiovas-
cular depression in the NTS [27].
However, the interactions between Ang II AT1 R/a2-adrenoreceptors seem to
be different [43]. The activation of a2-adrenergic receptors leads to an enhancement
of AT1 signaling, which is able to antagonize the recognition and signaling of
a2-adrenergic receptors, suggesting a homeostatic intramembrane inhibitory feed-
back mechanism (Fig. 9). However, Ang II AT1 receptors also antagonistically
interact with NPY Y1 and GAL receptor subtypes in a similar way. These multiple
receptor–receptor interactions may serve as homeostatic mechanisms in postulated
higher–order heteromers (receptor mosaics) to avoid too strong or too weak vaso-
depressor responses via the a2-adrenergic receptors; this could also exist as a
crucial hub receptor in higher-order heteromers of four or more different receptors
(a2-adrenergic receptor–GALR–NPYY1–AT1) (receptor mosaics). Thus, the net
Fig. 9 Schematic model illustrating the receptor interactions between Angiotensin II AT1
receptor subtype (AT1), NPY Y1 receptors subtype (Y1), NPY Y2 receptors subtype (Y2),
Galanin receptor (GALR) and Galanin receptor subtype preferring GAL(1–15) (GAL15-R) and
a2-adrenoreceptors (a2) in the NTS. These intramembrane receptor–receptor interactions may
take place in heterodimers and/or receptor mosaics of these different receptor subtypes. These
heteromers remain to be demonstrated and characterized in the NTS and dmnX. Positive signsindicate a synergistic interaction and negative signs indicate an antagonistic interaction. The
arrows show the direction of the interaction. Figure reproduced from [27] with permission
Neurochemical Modulation of Central Cardiovascular Control 127
cardiovascular response mediated by a2-adrenergic receptors is adjusted through
multiple allosteric receptor–receptor interactions mediated by postulated a2-adrenergic receptor- containing heterodimers and receptor mosaics involving also
Y1, AT1 and GAL receptor subtypes in the NTS and dmnX. These heterodimers
and receptor mosaics remain to be demonstrated and characterized in terms of
stoichiometry and topology and the demonstrated receptor–receptor interactions
described in this paper likely take place via allosteric mechanisms. A tetrameric
receptor mosaic, composed of AT1, GALR, Y1 and a2-adrenergic receptors, shouldalso be considered as a possibility when investigating the structural basis of several
of the demonstrated receptor–receptor interactions (Fig. 9).
Taken together, these results strengthen the role of GAL in central cardiovascu-
lar control and indicate the existence of receptor–receptor interactions among GAL
receptor subtypes as well as between GAL receptor subtypes and a2-adrenergicreceptors, and AT1 and Y1 receptor subtypes. These interactions are crucial in
understanding the integrative mechanisms responsible for the organization of the
cardiovascular responses from the NTS.
Acknowledgments This study was supported by Spanish DGCYT BFI2008-3369 and SEJ01323.
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