the emerging cardio-inhibitory role of the hippocampal...

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The emerging cardio-inhibitory role of the Hippocampal

Cholinergic Neurostimulating Peptide (HCNP)

Tommaso Angelone, Yannick Goumon, Maria Carmela Cerra, Marie-Hélène Metz-

Boutigue, Dominique Aunis, and Bruno Tota

Department of Pharmaco-Biology, University of Calabria, 87030 Arcavacata di Rende (CS) Italy

(T.A, M.C.C.); Department of Cell Biology, University of Calabria, 87030 Arcavacata di Rende

(CS) Italy (T.A., M.C.C., B.T.); Inserm U575 “Physiopatologie du Système Nerveux” 67084

Strasbourg- France (Y.G., M.-H. M.-B., D.A., T.A.)

JPET Fast Forward. Published on April 11, 2006 as DOI:10.1124/jpet.106.102103

Copyright 2006 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on April 11, 2006 as DOI: 10.1124/jpet.106.102103

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Running title: HCNP modulates cardiac function in the rat

Corresponding author

Bruno Tota

Department of Cell Biology - University of Calabria - 87030 Arcavacata di Rende (CS) Italy.

Phone. +39-0984-492907; Fax: +39-0984-492906 ; e-mail: [email protected]

Document statistics

Text pages: 25 Number of figures: 11 Number of references: 31 Abstract: 225 words Introduction: 722 words Discussion: 1175 words

ABBREVIATIONS

HCNP, Hippocampal Cholinergic Neurostimulating Peptide; PEBP, PhosphatidylEthanolamine-

Binding Protein; LVP, Left Ventricular Pressure; ACh, acetylcholine; ChoATase, choline

acetyltransferase enzyme; ISO, isoproterenol; PTx, Pertussis Toxin; RPP, rate pressure product; HR,

Heart Rate; +(LVdP/dt)max, maximal rate of left ventricular pressure contraction; CP, coronary

pressure.


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Abstract

Hippocampal Cholinergic Neurostimulating Peptide (HCNP), which derives from

PhosphatidylEthanolamine-Binding Protein (PEBP, also named Raf Kinase Inhibitor Protein,

RKIP), enhances ACh synthesis in the hippocampal medial septal nuclei. It is present in the

chromaffin secretory granules of the adrenal cells and under stress is co-secreted with peptide

hormones and catecholamines. Using the isolated rat heart perfused according to Langendorff to

reveal the cardiotropic action of HCNP on the mammalian heart, we showed that rat HCNP exerts,

at concentrations of 5x10-13 to 10-6 M, a negative inotropism under basal conditions (Left

Ventricular Pressure variations ranging from –8.34% ± 0.94 to –21% ± 3.5) and enhances the

cholinergic-mediated negative inotropy through direct interaction with G-protein-coupled

muscarinic receptor pathway. Under adrenergic stimulation (isoproterenol) the peptide exerts an

antiadrenergic action. The analysis of the percentage of Rate Pressure Product variations in terms of

EC50 values of isoproterenol alone (–8.5 ± 0.3; r2 = 0.90) and in the presence of rHCNP at 0.01 nM,

(–6.9 ± 0.36; r2= 0.88) revealed a competitive type of antagonism of the peptide. HCNP does not

affect either heart rate or coronary pressure. The evidence that HCNP in mammals may play a novel

role as an inhibitory cardiac modulator throughout an involvement of the myocardial G protein-

coupled receptor pathway provides new insights regarding the neurohumoral control of heart

function under normal and physiopathological conditions.


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Introduction

The “Hippocampal Cholinergic Neurostimulatory Peptide” (HCNP) is an undecapeptide named so

because it was initially purified from the rat hippocampus and enhanced acetylcholine (ACh)

synthesis of medial septal nuclei in an explant culture system (Ojika and Appel, 1984; Ojika et al.,

2000). In cholinergic neurons, HCNP exerts dose-dependent and time-dependent increases of both

activity and Vmax of choline acetyltransferase (ChoATase) (Ojika et al., 1994), inducing the key

enzyme expression required for ACh synthesis and influencing the development of cholinergic

phenotypes (Ojika et al., 1994). HCNP is the endogenous N-terminal fragment of the precursor

protein PhosphatidylEthanolamine-Binding Protein (PEBP), alternatively named Raf-1kinase

inhibitor protein (RKIP), since it interacts with cytoplasmic Raf1 (Yeung et al., 1999). PEBP is

ubiquitous, as documented by its occurrence in a wide range of organisms such as bacteria, yeasts,

plants, nematodes, drosophila and mammals (Schoentgen and Jolles, 1995). It has been shown,

particularly in mammals, that members of the PEBP family are involved in several signalling

systems, modulate the action of heterotrimeric G proteins and inhibit several serine proteases

(Hengst et al., 2001) as well as MAP kinase and NF-kB pathways (Vallèe et al., 2003). Studies on

the relationships between structural and physicochemical features of HCNP and its precursor

highlight an important dual function of this undecapeptide (Vallèe et al., 2003). First, HCNP is the

N-terminal positively charged region of PEBP and therefore it is able to interact with negatively

charged membranes by competition between intra-molecular and intermolecular electrostatic

interactions (Vallèe et al., 2003); at the same time, it participates in conformation and stability of the

active site of PEBP by hydrogen-bonding ability. Second, after its specific cleavage, free in

solution, HCNP appears well structured, at least in its central region (peptide sequence: D3LSKW7),

whose conformation is similar to that observed in crystallized PEBP (Vallèe et al., 2003). Therefore,

free HCNP is suited to act as a signal in target cells and tissues, thereby contributing to some of the

biological functions of PEBP (Hengst et al., 2001; Vallèe et al., 2003; Keller et al., 2004).


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Beyond their presence in the nervous tissue, both PEBP (e.g., adult rat: Frayne et al., 1999) and

HCNP (e.g., young rat: Katada et al., 1996) have been immunolocalized in other organs and tissues,

including blood vessel walls, adrenal gland, intestine and kidney. In addition, PEPB expression has

been detected on Western blots of heart and liver extracts (Frayne et al., 1999). Recently, an

estimation of PEBP concentration in bovine serum, obtained after protein sequencing, indicated a

value of 35 nM (Goumon et al., 2004). More recently, using chromaffin cells from bovine adrenal

medulla as a well characterized secretion model, we demonstrated that PEBP and HCNP are present

in chromaffin cells (intragranular matrix) from which they are secreted into the circulation with

catecholamines (Goumon et al., 2004). In addition, PEBP and HCNP have been detected with lipid

rafts, in platelet secretion and in serum (Goumon et al., 2004), in the cerebrospinal fluid of

Alzheimer patients (Tsugu et al., 1998) and in murine adipocytes (Kratchmarova et al., 2002). PEBP

was shown to be involved with several signalling cascades, interfering with MEK phosphorylation

and activation by Raf-1 (Yeung et al., 1999). Moreover, it was also postulated that PEBP is

involved in growth, transformation and differentiation, which are often deregulated in many forms

of cancer (Keller et al., 2004). Identified in the testis and epididymis, PEBP has been implicated in

spermatogenesis (Perry et al., 1994). These data, together with its multiple organ localization,

suggest that PEBP is a multifunctional protein with roles in reproduction, cardiology and neurology.

In order to have an insight of the functional features of HCNP as a signalling peptide, we have

recently used an in vitro frog working heart preparation as a bioassay system demonstrating that

HCNP from 10-11 to 10-7 M depresses the contractile performance, i.e. it exerts a negative inotropic

action, counteracting, at the same time, the classical positive inotropic effect of β-adrenergic

agonists (i.e. isoproterenol: ISO) (Goumon et al., 2004).

In the present paper, using the rat isolated and Langendorff perfused heart, a well-known paradigm

of cardiac physiology, we have provided evidence that rat HCNP counteracts the excitatory positive

inotropy of ISO. We have also shown that HCNP interacts with the cardiac ACh-signal transduction


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pathway, thus corroborating the idea that this undecapeptide may play a novel role as an inhibitory

cardiac modulator.

Methods

Animals

Male Wistar rats (Morini, Bologna-Italy S.p.A.) weighing 220 to 280 g were housed three per cage

in a ventilated cage rack system under standard conditions. Animals had food and water access ad

libitum. The investigation conforms to the Declaration of Helsinki and the Guide for the Care and

Use of Laboratory Animals published by US National Institutes of Health (NIH Publication No. 85-

23, revised 1996).

Isolated heart preparation

Rats were anaesthetized with intraperitoneal injection of ethyl carbamate (2 g/Kg body weight). The

hearts were rapidly excised and transferred in ice-cold buffered Krebs-Henseleit solution (KHs).

The aorta was immediately cannulated with a glass cannula and connected with the Langendorff

apparatus to start perfusion at a constant flow-rate of 12 ml/min (Cerra et al., 2005). Fluid

accumulation was prevented by piercing the apex of the left ventricle (LV). A water-filled latex

balloon, connected to a BLPR gauge (WRI, Inc. USA), was inserted through the mitral valve into

the LV to allow isovolumic contractions and continuously recording of mechanical parameters. The

balloon was progressively filled with water up to 80 µl to obtain an initial left ventricular end

diastolic pressure of 5-8 mmHg. Coronary pressure was recorded using another pressure transducer

located just above the aorta. The perfusion solution consisted of a modified non-recirculating Krebs-

Hanseleit solution (KHs) containing (in mM): NaCl 113; KCl 4.7; NaHCO3 25; MgSO4 1.2; CaCl2

1.8; KH2PO4 1.2; glucose, 11; mannitol 1.1; Na-pyruvate 5. pH was adjusted to 7.4 with NaOH and

the solution was equilibrated at 37°C with 95% O2 – 5% CO2. All drug-containing solutions were

freshly prepared before the experiments. ISO hydrochloride, carbachol, atropine, pirenzepine and


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PTx (Pertussis Toxin) were purchased by Sigma Chemical Company (St. Louis, MO, USA).

AFDX116 was a generous gift from Boehringer Ingelheim (Biberach, Germany). Rat HCNP

(rHCNP) was synthesized in “Inserm U575 Physiopathology of Nervous System”, Strasbourg-

France. Peptide synthesis - Acetylated rat HCNP was synthesized on an Applied Biosystems 433A

peptide synthesizer, using the stepwise solid-phase synthetic approach with 9-

fluorenylmethoxycarbonyl (Fmoc chemistry). A Na-acetyl-Alanine residue was used to obtain the

acetylated peptide. The peptide was further purified by reverse-phase high performance liquid

chromatography on a preparative Macherey-Nagel column, Nucleosil RP 300-7C18 (10 x 250 mm).

The peptide purity was checked by protein sequencing on an Applied Biosystems 473A protein

sequencer and MALDI-TOF mass spectrometry analysis (Biflex II, Bruker), showing a purity of

>95%. Haemodynamic parameters were assessed using a PowerLab data acquisition system and

analysed using Chart software (ADInstruments, Basile, Italy).

Experimental protocols

Basal conditions

Rat isolated and Langendorff perfused heart performance was evaluated by analysing heart rate

(HR, in beats/min), left ventricular pressure (LVP, in mmHg), rate pressure product (RPP: HR x

LVP, in 104 mmHg.beats/min), and +(LVdP/dt) max, i.e. the maximal rate of left ventricular

pressure. LVP and RPP were used as indexes of contractile activity and cardiac work (Georget et

al., 2003), respectively. The mean coronary pressure (CP, in mmHg) was calculated by averaging

the coronary pressure during several cardiac cycles.

Drugs application

HCNP stimulated preparations

To test desensitization we preliminarily perfused the heart with single repeated doses of HCNP at

10-10M and 10-7M revealing the absence of desensitization. In fact, each peptide dose produced a


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reduction of LVP of 16.6 ± 3.59% and 20.83 ± 5.09%, respectively. Thus, concentration-response

curves were generated by perfusing the cardiac preparations with KHs including increasing

concentrations of rHCNP (from 10-14 to 10-6 M) for 10 min.

Cholinergic stimulated preparations

To obtain preliminary information on the cholinergic action of rHCNP toward the carbachol-

dependent stimulation, dose-response curves were produced by perfusing the cardiac preparations

with KHs enriched with increasing concentrations of rHCNP (from 10-12 to 10-7 M) alone. These

curves were then compared with those obtained by exposing other cardiac preparations to the same

perfusion medium containing increasing concentrations of rHCNP (from 10-12 to 10-7 M) plus a

single concentration of carbachol (10 nM) or a single concentration of atropine (1 µM) or a single

concentration of AFDX116 (100 nM) or a single concentration of pirenzepine (1 nM). In all

protocols the hearts were perfused with KHs enriched with the specific drug before and after the

addition of HCNP. The antagonist concentration was selected on the basis of the results of

preliminary dose-response curves as the highest dose which did not significantly affect cardiac

performance.

Gi/O Protein involvement

To verify the involvement of Gi/O protein in the action mechanism of rHCNP, dose-response curves

were produced by perfusing the hearts with KHs enriched with increasing concentrations of rHCNP

(from 10-12 to 10-7 M) alone. These curves were then compared with those obtained by exposing

other cardiac preparations to the same perfusion medium containing increasing concentrations of

rHCNP (from 10-12 to 10-7 M) plus a single concentration of Pertussis Toxin (0.01 nM). As shown in

the rat heart, PTx catalyzes the ADP-ribosylation of the alpha-subunit of Gi/O and uncouples the

interaction between Gi and inhibitory receptors of adenylate cyclase, such as muscarinic receptors


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(Cohen-Armon and Sokolowsky, 1992; Grimm et al. 1998). The hearts were preincubated with PTx

for 60 min.

Isoproterenol stimulated preparations

In order to evaluate whether rHCNP antagonizes the ISO-stimulation, dose-response curves of ISO

(from 10-10 to 10-6 M) alone were compared to those obtained by exposing other cardiac

preparations to the same perfusion medium containing increasing concentrations of ISO (from 10-10

to 10-6 M) plus a single concentration of rHCNP (10-11 M).

Statistics

Data are expressed as the mean ± SEM. Since each heart represents its own control, the statistical

significance of differences within-group was assessed using paired Student’s t-test (p< 0.05).

Comparison between groups was made by using a one-way analysis of variance (ANOVA) followed

by Duncan and Bonferroni's Multiple Comparison Test. Differences were considered to be

statistically significant for p<0.05. The concentration-response curves of LVP reduction induced by

rHCNP alone and by rHCNP plus carbachol or atropine or AFDX116 or pirenzepine were fitted

using GraphPad Prism 4.02. This provided, for each curve, the –log of the concentration (in M)

which induced the 50% effect (EC50) of rHCNP alone and rHCNP plus carbachol, atropine,

AFDX116 and pirenzepine. The same fitting procedure was used to calculate the EC50 of ISO alone

and of ISO plus rHCNP on LVP and RPP.

Results

Isolated heart preparation

Basal conditions


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After the hearts were equilibrated for 20 minutes, HR was 280 ± 7 beats/min, LVP was 89 ± 3

(mmHg), RPP was 2.5 ± 0.1 104 mmHg beats/min, +(LVdp/dt)max was 2492 ± 129 (mmHg/s), CP

was 63 ± 3 mmHg. To assess the endurance and the stability of the preparation, the performance

variables were measured every 10 min showing that the heart was stable up to 180 min (Fig. 1A).

HCNP stimulated preparations

In the absence of desensitization, concentration-response curves of rHCNP were generated by

exposing the cardiac preparations to increasing concentrations of peptide. Peptide effects remained

stable up to 15 min, then gradually decreased with time. Accordingly, cardiac parameters were

measured at 10 min. rHCNP (from 5x10-13 to 10-6 M) caused a concentration-dependent negative

inotropic effect, revealed by a significant decrease of LVP (see trace in Fig. 1B), RPP and

(LVdP/dt)max (Fig. 2A), without modification of HR (Fig. 2B). Notably, rHCNP did not affect CP

(Fig. 2B).

Cholinergic stimulated preparations

To explore the interaction between rHCNP and intracardiac cholinergic signalling, hearts were

perfused with KHs containing Carbachol (10 nM), a non selective cholinergic agonist resistent to

the action of cholinesterases, in addition to increasing concentrations of rHCNP (from 10-12 to 10-7

M). Carbachol, at this concentration, did not modify LVP and RPP reductions induced by rHCNP.

In the presence of carbachol, rHCNP did not affect HR, except for a small significant decrease

observed at 10-10 and 10-9 M. At all concentrations, rHCNP plus carbachol induced a significant

increase of CP (Fig. 3). Atropine (1 µM), a competitive non-selective antagonist of muscarinic

receptors abolished the LVP reduction induced by rHCNP at 10-12, 10-8 and 10-7 M, while it reduced

these effects at 10-11, 10-10 and 10-9M. Atropine abolished or reduced the inhibitory effect of

rHCNP on RPP, depending on lower or higher peptide concentrations, respectively. Muscarinic

receptor blockade did not affect HR or CP (Fig. 4). The computerized fitting of the curves indicated


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the percentages of LVP variations obtained in the presence of rHCNP alone and rHCNP plus either

carbachol (10 nM) or atropine (1 µM) (Fig. 5). The sigmoid concentration-response curves and EC50

values (Fig. 5) showed a non-competitive antagonism of atropine on the rHCNP-mediated negative

inotropism.

To explore the involvement of different muscarinic receptor subtypes, rHCNP effects were

evaluated in the presence of a single concentration (100 nΜ) of either AFDX116 or pirenzepine,

competitive selective antagonists of M2- and M1-M3 subtype receptors, respectively. The treatment

with AFDX116 abolished the LVP reduction induced by rHCNP at 10-12, 10-11 and 10-10 M, while

significantly reducing it at 10-9, 10-8 and 10-7 M. AFDX116 abolished the inhibitory effect of

rHCNP on RPP at low peptide concentrations, without affecting it at higher peptide concentrations.

The treatment with the M2-receptors antagonist induced a HR reduction at high concentrations, and

a vasoconstriction from 10-10 to 10-7 M (Fig. 6).

The computerized fitting of the curves provides the percentage of variations of LVP obtained in the

presence of rHCNP alone and of rHCNP plus AFDX116 (100 nM). The resulting sigmoid

concentration-response curves and EC50 values (Fig. 8) indicate competitive antagonism of

AFDX116 on the rHCNP-mediated negative inotropism. In contrast, blockade of M1-M3 receptors

by pirenzepine was without significant effect on the rHCNP-induced cardiac effects (Fig. 7).

Gi/o Protein involvement

To verify the involvement of Gi/o protein system, cardiac preparations were perfused with KHs

containing PTx in presence of rHCNP. PTx abolished the rHCNP-mediated negative inotropism (i.e.

LVP and RPP) at all concentrations tested, while slightly decreasing HR at 10-9 and 10-8 M, and

increasing CP (Fig. 9). The representative trace reported in Fig. 1D shows the action of PTx on the

carbachol-induced LVP reduction.

Adrenergic stimulated preparations


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To test whether rHCNP is able to counteract the β-adrenergic-mediated positive chronotropism,

inotropism and coronary dilation, dose-response curves were generated by perfusing heart

preparations with increasing concentrations of ISO (from 10-10 to 10-6 M) alone and together with a

single concentration of rHCNP (0.01 nM). rHCNP abolished the ISO (from 5x10-9 M to 10-7 M)-

mediated positive increase of LVP and RPP. rHCNP reduced HR at low ISO concentrations (up to

50nM) while increased HR at high ISO concentrations (0.5 and 1µM). CP was not modified (Fig.

10). Figure 1C shows a representative trace of HCNP 0.01 nM effects in the presence of a single

ISO concentration (5 nM).

The antagonistic effect of rHCNP against the ISO-dependent stimulation, evaluated by fitting the

sigmoid concentration–response curves of LVP and RPP in the presence of ISO alone (from 10-10 to

10-6 M) and ISO (from 10-10 to 10-6 M) plus rHCNP (0.01 nM), provided the corresponding

percentage of LVP variations in terms of EC50 values. These data are consistent with a competitive

type of antagonism of rHCNP (Fig. 11 A and B).

Discussion

To date, recent data on PBEP and its derived HCNP implicate an involvement in multiple signalling

mechanisms. In the present study we show the involvement of HCNP in the myocardial G protein-

coupled receptor pathways acting as negative modulator of the adrenergic signalling. In fact, using

the model of isolated and Langendorff perfused rat heart, we demonstrate, for the first time in

mammals, that HCNP exerts direct inotropic effects, providing also clear evidence that these

cardiotropic actions involve interaction with receptor-mediated heterotrimeric G protein signalling.

These data confirm our preliminary study showing that HCNP depresses the mechanical

performance of the isolated and perfused working heart of the frog by inducing negative inotropism

under basal conditions and counteracting the ISO-dependent positive inotropism (Goumon et al.,

2004).


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Basal conditions- Under basal conditions (i.e. non-stimulated), HCNP appears to act as an efficient

negative modulator of myocardial performance, since it depresses relevant contractile parameters,

such as LVP and RPP, within a nanomolar range of concentrations. Significant effects were elicited

by HCNP at the same concentrations of its precursor, PEPB, found in the bovine serum (Goumon et

al, 2004). Notably, the concentration/response curves show that neither HR nor CP is modified by

HCNP, highlighting a selective inotropic characteristic of the peptide. This feature makes it of

particular interest for physio-pharmacological studies designed to analyze myocardial contractility

separately from rhythmogenic or coronary motility.

Cholinergic signalling - The findings that HCNP-dependent negative inotropism is enhanced in the

presence of cholinergic stimuli (i.e. carbachol) and is abolished in the presence of non specific

muscarinic inhibition (i.e. atropine) demonstrate the undecapeptide involvement in the cardiac

cholinergic signal-transduction pathway. Moreover, using selective muscarinic subtype inhibitors,

we provide evidence that HCNP acts as a functional agonist of ACh at the M2 subtype receptors

level, without any significant interaction with the M1-M3 receptor subtypes. In the human heart, M2

subtype is preferentially located on the myocardiocytes (Fu et al., 1994). In the rat heart M2

muscarinic receptors were detected mainly on the sarcolemma and T-tubules of the ventricular

cardiomyocytes (Hove-Madsen et al., 1996). They are principally coupled to adenylate cyclase

inhibition, reduce intracellular cAMP levels and decrease the L-type Ca2+ current, thereby eliciting

negative cholinergic chronotropic and inotropic effects (for references, see Hove-Madsen et al.,

1996; Gattuso et al., 1999). However, it cannot be excluded that in other tissues, e.g. endothelial

cells, HCNP may exert functional effects via interaction with M1-M3 receptors. A functional

heterogeneity of cardiac muscarinic receptors has been reported in the human heart. It has been

shown that they exert physiological effects considerably different between atrium and ventricle,

these differences being attributed to coupling with different G proteins and downstream cascades

(Mittmann et al., 2003). It could be that HCNP interacts with ventricular muscarinic receptors

differently from how it interacts with the receptors expressed in the conduction system. This may


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contribute to explain why HCNP did not exert chronotropic effects. The finding that, on the bases of

EC50 values AFDX116 exerts competitive inhibition on HCNP-mediated negative inotropism, while

atropine appears to act in a non competitive manner, cannot be explained by the present study. In

the absence of a demonstration of a direct interaction of HCNP with the receptor binding site,

alternative explanations (e.g. allosteric modulation) should be taken into consideration. In the

Langendorff heart preparation the sympathetic postganglionic fibers are denervated because they are

separated from the ganglion cell bodies in the stellate ganglia, while parasympathetic preganglionic

fibers are transected decentralizing the heart. This allowed us to evaluate HCNP effects without

parasympathetic and sympathetic extrinsic influences. However, since the rat ventricles exhibit a

high cholinergic nerve density, an intracardiac release of ACh by the peripheral nerve endings

cannot be excluded. Cholinergic-mediated negative inotropism can also be elicited by activation of

constitutive NOS activity (eNOS) linked to the muscarinic receptor-G protein system detected in the

caveolae of ventricular myocytes of several mammalian hearts (for references, see Dessy et al.,

2000). Our results are consistent with an endothelium-independent inotropic action of HCNP,

indicating that the peptide directly targets the myocardium. Remarkably, the HCNP-mediated

inotropism is G protein dependent. Using the precursor of HCNP, i.e. the human PEBP (hPEBP), it

has been shown that hPEBP modulates G protein and G protein-coupled receptor signalling

(Kroslak et al., 2001).

Receptor-receptor interactions between G-protein-coupled receptors occurring at the plasma-

membrane level have been identified. In particular, it has been shown that clustering of G-protein-

coupled receptors in aggregates or receptor mosaics results in the reciprocal modulation of their

binding and decoding characteristics, their cooperativity playing an important part in the decoding

of signal transduction (Agnati et al., 2005).

The analysis of the interactions between HCNP and the adrenergic system has revealed that the

peptide exerts a functional competitive antagonism. This was shown by the right shift of the

sigmoid curves of LVP and RPP. HCNP modified ISO-dependent chronotropism with a biphasic


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effect. In fact, HCNP reduced the positive chronotropic effect of low ISO concentrations while it

increased the ISO effect at higher concentrations. Presently, we cannot explain the mechanisms

underlying the biphasic effect of HCNP on ISO-dependent positive chronotropism. Changes in the

ratio of intracellular modulators (i.e. cAMP/cGMP), which may be related to this effect, can be

postulated (Casadei and Sears, 2003, and references therein). Taken together, these data suggest that

by mimicking ACh, HCNP in the rat heart may act as a new cholinergic modulator through direct

interaction with the G-protein-muscarinic coupled receptor signal-transduction system localized at

the myocardiocyte sarcolemmal level. HCNP-induced activation of the cholinergic pathway appears

to counteract functionally the adrenergic pathway, thereby modulating myocardial contractility

under basal conditions and under adrenergic stimulations.

This putative role of HCNP and its precursor PEBP may provide a new breakthrough regarding the

important functional link between the brain and the cardiovascular system, achieved through two

pathways, i.e. the autonomic nervous system (ANS) acting via direct neural actions, and the

neuroendocrine humoral outflow acting via the hypothalamic-pituitary-adrenal (HPA) axis. Since

the 30’s, Walter Cannon studying the stress response, by him named as “fight or flight” reaction,

recognized the “wisdom of the body” in the “generalized” sympathetic response acting during

stress, and emphasized, at the same time, the more “discrete” influences exerted by the

parasympathetic nervous system (PNS) (Chrousos and Gold, 1992). The heart, being an integrative

interface between the noradrenergic nerve terminals, releasing noradrenaline, and the circulating

catecholamines (CAs) secreted by the adrenal medulla, represents a classical paradigm of a stress-

threatened organ. Elevated CAs levels have long been known to induce necrotic heart damage, since

the pioneer work of Raab (Raab, 1963; Tan et al., 2003; Teerlink et al., 1994; and references

therein). Knowledge on HCNP and cardiac signalling may provide a new molecular dimension to

the “wisdom of the body”, uncovering how the sympathetic and parasympathetic systems influence

effector tissues homeostasis and, at the same time, how over-activation of the adrenergic system

during the onset and course of patho-physiological conditions may be functionally counteracted.


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In conclusion, our present study strongly suggests that HCNP and the precursor protein PEBP are

new endocrine factors involved in the control of cardiac function physiology in mammals.


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Footnotes

This work was supported by MURST 60%: B. Tota and M.C. Cerra, the “Dottorato di Ricerca in

Biologia Animale” (T. Angelone) and the Egide Program (Galilée, B.Tota, T. Angelone, M.C.

Cerra) and Vinci Program (T.Angelone). Thanks are given to Laura Jean Carbonaro for revising the

text.

Person receiving reprint request

Bruno Tota

Department of Cell Biology - University of Calabria - 87030 Arcavacata di Rende (CS) Italy.

Phone. +39-0984-492907; Fax: +39-0984-492906 ; e-mail: [email protected]

1Tommaso Angelone, 2Yannick Goumon, 3Maria Carmela Cerra, 4Marie-Hélène Metz-Boutigue,

5Dominique Aunis, and 6Bruno Tota


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Legends for Figures

Fig. 1. Representative LVP (Basal value= 89 ± 3 mmHg) traces showing (A) the time course

obtained in the presence of vehicle alone; (B) the effects of increasing concentrations (10−14 to 10−6

M) of HCNP (each arrow represents the administration of a single concentration of the peptide); (C)

the effect of ISO alone (5 nM) and in the presence of HCNP (0.01 nM); (D) the effect of Carbachol

alone (10 nM) and in presence of PTx (0.01 nM).

Fig 2. Concentration-dependent responses of rHCNP (10−14 to 10−6 M) on LVP (left ventricular

pressure), RPP (rate pressure product), (LVdP/dt)max (maximal rate of left ventricular pressure)

(A) and CP (coronary pressure), HR (heart rate) (B), on the isolated and Langendorff perfused rat

heart preparation. Basal values: HR=280 ± 7 beats/min, LVP=89 ± 3 (mmHg), RPP=2.5 ± 0.1 104

mmHg beats/min, +(LVdp/dt)max= 2492 ± 129 (mmHg/s), CP=63 ± 3 mmHg. Percentage changes

were evaluated as means ± SEM of 13 experiments. Significance of difference from control values

(t-test);*=p<0.05. Comparison between groups (ANOVA, Duncan's test); §=p<0.05.

Fig 3. Concentration-dependent responses of rHCNP alone (10-12 ÷ 10-7 M) and rHCNP (from 10-12

to 10-7 M) plus Carbachol (10 nM) on HR, LVP, RPP and CP on the isolated and Langendorff

perfused rat heart preparation. Basal values: HR=280 ± 7 beats/min, LVP=89 ± 3 (mmHg),

RPP=2.5 ± 0.1 104 mmHg beats/min, CP=63 ± 3 mmHg. Percentage changes were evaluated as

means ± SEM of 7 experiments for each group. Significance of difference from control values (t-

test); *,+=p<0.05.


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Fig 4. Concentration-dependent responses of rHCNP alone (from 10-12 to 10-7 M) and of rHCNP

(from 10-12 to 10-7 M) plus atropine (1 µM) on HR, LVP, RPP and CP on the isolated and

Langendorff perfused rat heart preparation. Basal values: HR=280 ± 7 beats/min, LVP=89 ± 3

(mmHg), RPP=2.5 ± 0.1 104 mmHg beats/min, CP=63 ± 3 mmHg. Percentage changes were

evaluated as means ± SEM of 7 experiments for each group. Significance of difference from control

values (t-test); *,+=p<0.05. Comparison between groups (ANOVA, Duncan's test); §=p<0.05.

Fig 5. The sigmoid concentration-response curves of rHCNP-mediated inhibition on LVP of rHCNP

alone (from 10-12 to 10-7 M) and of rHCNP (from 10-12 to 10-7 M) plus a single concentration of

carbachol (10 nM) or atropine (1 µM) on the isolated and Langendorff perfused rat heart. Inhibition

of contractility is expressed as a percentage of LVP [baseline=0%, peak inhibition by rHCNP and

rHCNP plus Carbachol or Atropine=-100%].

The EC50 value (in logM) of rHCNP alone was -12.05 ± 0.15 (r2= 0.97), of rHCNP plus carbachol

(10 nM) was -11.74 ± 0.28 (r2= 0.91), of rHCNP plus atropine (1 µM) was -11.90 ± 0.77 (r2= 0.64).

Comparison between groups (ANOVA, Duncan’s and Bonferroni's Multiple Comparison Test);

§=p<0.05.

Fig 6. Concentration-dependent responses of rHCNP alone (from 10-12 to 10-7 M) and of rHCNP

(from 10-12 to 10-7 M) plus AFDX116 (100 nM) on HR, LVP, RPP and CP on the isolated and

Langendorff perfused rat heart. Basal values: HR=280 ± 7 beats/min, LVP=89 ± 3 (mmHg),



test); *,+=p<0.05. Comparison between groups (ANOVA, Duncan's test); §=p<0.05.

Fig 7. Concentration-dependent responses of rHCNP alone (10-12 to 10-7 M) and of rHCNP (from

10-12 to 10-7 M) plus pirenzepine (1 nM) on HR, LVP, RPP and CP on the isolated and Langendorff


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test); *,+=p<0.05.

Fig 8. The sigmoid concentration-response curves of rHCNP-mediated inhibition on LVP of rHCNP

alone (from 10-12 to 10-7 M) and of rHCNP (from 10-12 to 10-7 M) plus a single concentration of

AFDX116 (100 nM) or a single concentration of pirenzepine (1 nM) on the isolated and

Langendorff perfused rat heart preparation. Inhibition of contractility is expressed as a percentage of

LVP [baseline=0%, peak inhibition by rHCNP and rHCNP plus AFDX116 or rHCNP plus

pirenzepine= -100%].

The EC50 value (in logM) of rHCNP alone was -12.05 ± 0.15 (r2= 0.97), of rHCNP plus AFDX116

(100 nM) was -8.17 ± 0.38 (r2= 0.88), of rHCNP plus pirenzepine (1 nM) was 10.42 ± 0.23 (r2=

0.96). Comparison between groups (ANOVA, Duncan’s Multiple Comparison Test); §=p<0.05.

Fig 9. Concentration-dependent responses of rHCNP alone (from 10-12 to 10-7 M) and rHCNP (from

10-12 to 10-7 M) plus PTx (0.01 nM) on HR, LVP, RPP and CP on the isolated and Langendorff




test); *=p<0.05. Comparison between groups (ANOVA, Duncan's test); §=p<0.05.

Fig 10. Concentration-dependent responses of ISO alone (from 10-10 to 10-6 M) and of ISO (from

10-10 to 10-6 M) plus rHCNP (0.01 nM) on HR, LVP, RPP and CP on the isolated and Langendorff




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test); *=p<0.05. Comparison between groups (ANOVA, Duncan's test); §=p<0.05.

Fig 11A,B. The sigmoid concentration-response curves of ISO-mediated stimulation on LVP and

RPP of ISO (from 10-10 to 10-6 M) alone and ISO (from 10-10 to 10-6 M) plus a single concentration

of rHCNP at 0.01 nM on the isolated and Langendorff perfused rat heart preparation. Contraction is

expressed as a percentage of LVP and RPP [baseline=0%, peak constriction by ISO and ISO plus

VS-1=100%].

The EC50 value (in logM) of ISO alone was -8.5 ± 0.3 (r2= 0.90), of ISO plus HCNP (0.01 nM) was

-6.9 ± 0.36 (r2= 0.88). Comparison between groups (ANOVA, Duncan's test); §=p<0.05.


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