Neurochemical Modulation of Central Cardiovascular Control: The Integrative Role of Galanin

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

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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


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


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

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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


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


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.


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].


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


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


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


a

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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

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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


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


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


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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Neurochemical Modulation of Central Cardiovascular Control: The Integrative Role of Galanin

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