General and Comparative Endocrinology 171 (2011) 245–251

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General and Comparative Endocrinology

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Central pituitary adenylate cyclase-activating polypeptide (PACAP) andvasoactive intestinal peptide (VIP) decrease the baroreflex sensitivity in trout

Frédéric Lancien a, Nagi Mimassi a, J. Michael Conlon b, Jean-Claude Le Mével a,⇑a Université Européenne de Bretagne, Université de Brest, INSERM U650, Laboratoire de Traitement de l’Information Médicale, Laboratoire de Neurophysiologie,IFR 148 ScInBioS, Faculté de Médecine et des Sciences de la Santé, 22 Avenue Camille Desmoulins, CS 93837, 29238 Brest Cedex 3, CHU de Brest, Franceb Department of Biochemistry, Faculty of Medicine and Health Sciences, United Arab Emirates University, 17666 Al Ain, United Arab Emirates

a r t i c l e i n f o

Article history:Received 6 October 2010Revised 1 February 2011Accepted 9 February 2011Available online 12 February 2011

Keywords:PACAPVIPR–R interval and systolic blood pressurevariabilitiesBaroreflexIntracerebroventricular injectionIntra-arterial injectionTransfer function analysisTeleost

0016-6480/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.ygcen.2011.02.006

Abbreviations: BRS, baroreflex sensitivity; ECG,frequency; HRV, heart rate variability; IA, intra-arterular; LF, low frequency; NPO, preoptic nucleus; PACAPactivating polypeptide; PAC1, PACAP receptor; PDA,PSD, power spectral density; PwO2, partial oxygen prblood pressure; VIP, vasoactive intestinal peptide;subtype 1; VPAC2, VIP/PACAP receptor subtype 2.⇑ Corresponding author. Fax: +33 2 9801 6474.

E-mail address: jean-claude.lemevel@univ-brest.fr

a b s t r a c t

Although PACAP and VIP exert diverse actions on heart and blood vessels along the vertebrate phylum, noinformation is currently available concerning the potential role of these peptides on the regulation of thebaroreflex response, a major mechanism for blood pressure homeostasis. Consequently, the goal of thisstudy was to examine in our experimental model, the unanesthetized rainbow trout Oncorhynchusmykiss, whether PACAP and VIP are involved in the regulation of the cardiac baroreflex sensitivity(BRS). Cross-spectral analysis techniques using a fast Fourier transform algorithm were employed to cal-culate the coherence, phase and gain of the transfer function between spontaneous fluctuations of sys-tolic arterial blood pressure and R–R intervals of the electrocardiogram. The BRS was estimated as themean of the gain of the transfer function when the coherence between the two signals was high andthe phase negative. Compared with vehicle, intracerebroventricular (ICV) injections of trout PACAP-27and trout VIP (25–100 pmol) dose-dependently reduced the cardiac BRS to the same extent with a thresh-old dose of 50 pmol for a significant effect. When injected intra-arterially at the same doses as for ICVinjections, only the highest dose of VIP (100 pmol) significantly attenuated the BRS. These results suggestthat the endogenous peptides PACAP and VIP might be implicated in the central control of cardiacbaroreflex functions in trout.

� 2011 Elsevier Inc. All rights reserved.

1. Introduction

Pituitary adenylate cyclase-activating polypeptide (PACAP) wasoriginally isolated from ovine hypothalamus on the basis of itsability to stimulate adenylate cyclase activity in adenohypophysialcells [28]. PACAP is found in two biologically active forms, a 38amino-acid peptide (PACAP-38) and a C-terminally truncated 27amino-acid peptide (PACAP-27). In humans, the N-terminal por-tion of PACAP shows 68% sequence identity with vasoactive intes-tinal peptide (VIP), identifying PACAP as a member of the VIP/secretin/glucagon/GH-releasing hormone superfamily of peptides[39]. PACAP and VIP exert their biological activities through threeG-protein coupled receptors termed PAC1, VPAC1 and VPAC2

ll rights reserved.

electrocardiogram; HF, highial; ICV, intracerebroventric-, pituitary adenylate cyclase-

dorsal aortic blood pressure;essure in water; SBP, systolicVPAC1, VIP/PACAP receptor

(J.-C. Le Mével).

[20]. Consistent with the wide distribution of PACAP and VIP, andalso their receptors, throughout the central nervous system andperiphery, PACAP and VIP exert multiple actions [44]. On theperipheral cardiovascular system, PACAP and VIP are consideredto have potent and direct vasodilatory properties on a variety ofarterial blood vessels and also to exert stimulatory effects on theheart [14,44]. Nonetheless, the role of these peptides in central car-diovascular regulation is not so clearly delineated [14]. In particu-lar, nothing is known about the possible action of central PACAPand VIP on baroreflex, a key mechanism for blood pressurehomeostasis.

The baroreflex has been evolutionary conserved from Agnatha(lamprey) to humans [40,19]. The baroreflex in fish, as in humans,is working spontaneously under baseline conditions [22] and re-sponds to adverse blood pressure changes [40,38]. This baroreflexresponse is probably used as a mechanism to protect the delicatevasculature of the fish gills against high blood pressure [3,40].Additionally, the biologically active region of PACAP, (the N-termi-nal 27 amino acids), the sequence of VIP, and the primary structureof PACAP/VIP receptors have been remarkably well conserved fromfish to humans [45,39,32]. In teleost fish, the PACAP/VIP system iswidely distributed in peripheral tissues [35,18,10] and in the

246 F. Lancien et al. / General and Comparative Endocrinology 171 (2011) 245–251

central nervous system [29]. We recently described the cardiore-spiratory actions of these peptides after peripheral and centraladministration in trout [23]. After intracerebroventricular (ICV)injection, PACAP and to lesser extend VIP provoked an hypervent-ilatory effect but only PACAP produced an increase in mean dorsalaortic blood pressure (PDA) without changing heart rate. Intra-arte-rial (IA) injections of either PACAP or VIP were without effect onventilation and only VIP significantly elevated PDA without chang-ing heart rate. The lack of heart rate response to elevation of bloodpressure suggests that the cardiac baroreflex sensitivity (BRS) maybe depressed following central PACAP and IA VIP. Therefore, theaim of this study was to examine in our experimental model, theunanesthetized rainbow trout Oncorhynchus mykiss, whether PA-CAP and VIP are involved in the regulation of the BRS. For this pur-pose, trout PACAP and VIP were injected within the third ventricleof the brain and intra-arterially and the modern transfer functionanalysis technique was used to study the cardiac BRS.

2. Materials and methods

2.1. Peptides and chemicals

Trout PACAP-27 (HSDGIFTDSYSRYRKQMAVKKYLAAVL.NH2)and trout VIP (HSDAIFTDNYSRFRKQMAVKKYLNSVLT.NH2) weresynthesized by GL Biochem (Shanghai, China) and purified to nearhomogeneity (>98% purity) by reversed-phase HPLC. The identitiesof the peptides were confirmed by electrospray mass spectrome-try. Peptides were stored in stock solution (0.01% HCl) at �25 �C.For injections, the peptides were diluted to the desired concentra-tion with Ringer’s solution (vehicle) immediately prior to use. Thecomposition of the Ringer’s solution was (in mM): NaCl 124, KCl 3,CaCl2 0.75, MgSO4 1.30, KH2PO4 1.24, NaHCO3 12, glucose 10 (pH:7.8). All solutions were sterilized by filtration through 0.22 lm fil-ters (Millipore, Molsheim, France) before injection.

2.2. Animals and surgical procedures

Some of the results reported in the present paper refer torecordings made during our own previous study on the action ofPACAP and VIP in trout [23]. Recordings were excluded from theanalysis if they contained excessive artefacts on electrocardiogram(ECG) signal or on pulsatile blood pressure. Additional new exper-iments were also carried out on rainbow trout O. mykiss usingexperimental procedures that have been described in detail in pre-vious work [23]. Briefly, rainbow trout (body wt. 240–270 g) wereequipped with two electrocardiographic electrodes, a dorsal can-nula, and an ICV microguide with a buccal catheter that was usedto record the buccal ventilatory pressure (not quantified in thepresent study). After surgery, the animals were transferred to a6 l blackened chamber supplied with dechlorinated and aeratedtap water (10–11 �C) that was both recirculating and through-flowing. Oxygen pressure within the water tank (PwO2) and pHwere continuously recorded and maintained at constant levels(PwO2 = 20 kPa; pH = 7.4–7.6). The trout were allowed to recoverfrom surgery and to become accustomed to their new environmentfor 48–72 h. Experimental protocols were approved by the Regio-nal Ethics Committee in Animal Experiments of Brittany, France.

2.3. Intracerebroventricular and intra-arterial administrations ofPACAP and VIP

For all protocols, the recording session lasted 30 min and allinjections were made at the fifth minute of the test. For the ICVprotocol, the fish received an ICV injection of vehicle (0.5 ll) oran ICV injection of trout PACAP or VIP (25, 50 and 100 pmol in

0.5 ll). For the IA protocol, 50 ll of vehicle, trout PACAP or VIP atdoses of 25, 50 and 100 pmol was injected through the dorsal aortacannula and immediately flushed by 150 ll of vehicle. Peptideswere administered in random order.

2.4. Data acquisition and transfer function analysis of thecardiovascular variables

The ECG and PDA signals were recorded using standardized elec-tronic devices [23]. The output signals were digitalized at 1000 Hz,visualized on the screen of a PC during the 30 min recording periodand finally stored using PowerLab 4/30 data acquisition system(ADInstruments, Oxford, England) and LabChart Pro software(v.6.0; ADInstruments, Oxford, England). ECG and PDA signals wereprocessed off-line with custom-made programs written in LabView6.1 (Laboratory Virtual Instrument Engineering Workbench, Na-tional Instruments, Austin, USA). For all protocols, 10 min seg-ments of ECG and PDA signals were selected 15 min after ICV orIA injections of vehicle, PACAP or VIP. R–R intervals were deter-mined after detection of the R waves from the ECG recordingsand systolic blood pressure (SBP) was identified from the pulsatilePDA. Their mean values were calculated. R–R interval and SBP timeseries were resampled at 2.56 Hz to obtain equidistant data points.The linear trend was removed from this new time series and 11segments of 256 data points (100 s) overlapping by half were sub-jected to a Hanning window. Spectral and cross spectral techniquesdeveloped in the present study were adapted from methods de-scribed previously in human [9,34], rat [16] and lizard [12]. Thepower spectral density (PSD) of each segment was calculated usingstandard fast Fourier transform and the PSD spectrum obtainedwere averaged. In order to investigate to what extent the input sig-nal (the SBP) influences the output signal (the R–R interval) thecoherence, phase and transfer function spectra of SBP against R–R interval were determined. The coherence spectrum, which hasvalues between 0 and 1, is a measure of the correlation betweenthe variations of the two signals. The transfer function provides ameasure of the degree to which input signal content, at a given fre-quency, appears in output energy. A high coherence (>0.5) betweenthe two signals and a negative phase shift indicates that the SBPmediates the changes in R–R intervals. Consequently, the cardiacBRS was estimated as the mean of the gain of the transfer functionwhen the coherence was high and the phase negative.

All calculations for R–R interval (in msec), SBP (in kPa), PSD (inkPa2/Hz), coherence, phase function (in sec) and transfer gain (inkPa/msec) were made for the post-injection period of 20–30 minand the results were averaged for trout subjected to the sameprotocol.

2.5. Statistical analysis

Data are expressed as means ± SEM or +SEM. In the figures andtable, data refer to absolute values. For comparison betweengroups, Kruskal–Wallis non-parametric one-way analysis of vari-ance followed by Dunn’s multiple comparison test was used. A va-lue of P < 0.05 was considered significant. The statistical tests wereperformed and the graphs constructed using GraphPad Prism 5.0(GraphPad, San Diego, CA).

3. Results

3.1. Baroreflex response to central PACAP

Fig. 1 shows a representative example of 5 min R–R intervaltime-series, and SBP time series recorded during the 20–30 minperiod in a trout receiving firstly an ICV injection of vehicle

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F. Lancien et al. / General and Comparative Endocrinology 171 (2011) 245–251 247

(Fig. 1A) followed 30 min later by an ICV injection of 50 pmolPACAP (Fig. 1B). Comparing the PACAP-injected trout with thevehicle-injected trout, PACAP did not produce any obvious changein the mean R–R interval value but decreased the R–R intervalvariability. Moreover, PACAP provoked an increase in SBP and fol-lowing the injection, the SBP time-series showed more clearlyshaped and enhanced low frequency oscillations. Fig. 2 showsan example of the average results obtained during the differentsteps of the transfer function analysis of 10 min R–R intervaland SBP time-series for all trout receiving an ICV injection ofvehicle or PACAP (50 pmol). For the PSD of the R–R interval var-iability, two main frequency peaks appeared: a low frequency (LF)component in the 0–0.1 Hz frequency band and a high frequency(HF) peak located between 0.1 and 0.2 Hz and exhibiting thehighest PSD (Fig. 2A). For the PSD of the SBP variability, a mainLF component appeared in the 0–0.1 Hz frequency band peakingat 0.04 Hz followed by a minor HF component in the 0.1–0.2 Hzfrequency range (Fig. 2B). The ICV injection of PACAP decreasedthe PSD of the HF peak of the R–R interval variability (Fig. 2A)but amplified the LF peak (Fig. 2B). After PACAP the PSD of theSBP-LF band increased (Fig. 2B). Following ICV injection of vehicleor PACAP a high coherence (>0.5) between the two signals wasobserved within the 0.1–0.2 Hz frequency range (Fig. 2C). Thenegative phase between the two signals in the 0.1–0.2 Hz fre-quency band after ICV vehicle or PACAP indicates that the inputsignal (the SBP) drives the output signal (the R–R interval)(Fig. 2D). The transfer gain, giving an estimate of the BRS, showsthat the BRS in the 0.1–0.2 Hz frequency band is decreased fol-lowing PACAP injection compared to vehicle-injected trout(Fig. 2E). The histograms in Fig. 3 summarize the average changesin R–R interval, SBP and in the transfer function gain between SBPand R–R interval spectra after ICV injection of vehicle or a rangeof doses (25–100 pmol) of PACAP. Compared with vehicle-in-jected trout, PACAP produced a dose-dependent increase in SBPbut no change in R–R intervals (Fig. 3A). Fig. 3B demonstrates thatthe transfer function gain, i.e. the BRS, is dose-dependently de-creased following ICV PACAP.

3.2. Baroreflex response to central VIP

The effect of VIP on the cardiovascular variables and cardiac BRSare summarized in Fig. 4. In contrast to PACAP, the ICV injection ofVIP (25–100 pmol) elicited a non-significant increase in SBP and anon-significant decrease in R–R intervals (Fig. 4A). Nonetheless,ICV VIP produced a significant and dose-dependent decrease inthe gain of the transfer function compared to vehicle-injected trout(Fig. 4B), a response that is identical to that observed following ICVPACAP (Fig. 3B). Consequently, the cardiac BRS is also decreasedfollowing central VIP.

3.3. Baroreflex response to peripheral PACAP and VIP

Similar computerized transfer function analyses of the cardio-vascular variables were performed after peripheral administrationof vehicle, PACAP and VIP. After each treatment, the coherence be-tween the PSD of the R–R interval and the SBP variabilities washigh and the phase was negative within the 0.1–0.2 Hz frequencyband (not shown). Table 1 summarizes all changes observed inthe cardiovascular variables, and transfer function gain after thevarious treatments. Peripheral PACAP did not produce any signifi-cant change in the R–R interval and SBP values compared to vehicleinjection and, in contrast to central PACAP, the BRS is not influ-enced by peripheral PACAP (Table 1). Table 1 also shows that onlythe highest dose of peripheral VIP (100 pmol) significantly in-creased the SBP and non-significantly reduced the R–R intervalscompared to the peripheral administration of vehicle. Moreover,at this high dose, VIP significantly reduced the transfer functiongain compared to vehicle injection (Table 1).

4. Discussion

To our knowledge, our results demonstrate for the first time inany vertebrate class that exogenous administration of the two neu-rohormonal peptides PACAP and VIP in the brain reduces the

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248 F. Lancien et al. / General and Comparative Endocrinology 171 (2011) 245–251

cardiac BRS. The effects of the two peptides are most probablymediated primarily by the central nervous system since peripheralinjection of VIP reduced the BRS only at its highest dose. The re-sults have been obtained using the modern signal processing tech-nique, transfer function analysis, that allows determination of thespontaneous cardiac baroreflex from cross-spectral analysis ofthe variabilities in R–R intervals of the ECG and SBP [9,16,34]. Thistechnique obviates the need to use of intra-arterial administrationof hypertensive and hypotensive pharmacological drugs to evokereciprocal cardiac baroreflex responses. These substances may dif-fuse to the CNS and may produce confounding effects on the baro-reflex responses. As well as providing an appropriate method toestimate the gain of the spontaneous cardiac baroreflex, transferfunction analysis also offers during its various steps an attractivemethod to analyze the rhythmic oscillations spontaneously occur-ring in R–R intervals and SBP [34]. This is exemplified in Fig. 2 ofthe present work and we will focus the discussion on the charac-teristics of the transfer function analysis after ICV injection of vehi-cle and PACAP since the action of VIP was virtually identical to thatof PACAP.

The spectral analysis of R–R interval variability, also called theheart rate variability (HRV), after ICV injection of vehicle gave a re-sult which is consistent with previous studies in trout [11,25] anddemonstrated that HRV had two fundamental components: a LFcomponent within the 0–0.1 Hz frequency band and a HF compo-nent within the 0.1–0.2 Hz frequency band. Cardiac vagotomy oratropine administration abolished HRV in teleost fish [1,25,6]while propanolol injection was without significant action on HRV[1] demonstrating that the vagal parasympathetic nervous systemis the main, or even the exclusive, contributor to HRV in teleost fish[15]. After ICV injection, PACAP decreased the R–R interval vari-ability suggesting that PACAP reduced the cardiac vagal outflowfrom the brainstem. To the best of our knowledge, spectral analysisof SBP had never been described in fish. The LF component of theSBP variability which peaked between 0.03 and 0.04 Hz (25–33 speriod; Fig. 1B) suggests that the oscillations in the SBP might cor-respond to Mayer waves. Mayer waves have already been de-scribed in trout [46,24]. Mayer waves in humans have acharacteristic frequency of about 0.1 Hz [43] and 0.4 Hz in rats[5]. It is generally accepted that arterial pressure Mayer waves in

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Fig. 4. Histograms showing (A) the R–R intervals (scale on the left) and the SBPvalues (scale on the right), (B) the BRS during the 15–25 min period afterintracerebroventricular injection of 0 (vehicle, n = 11), 25 pmol (n = 6), 50 pmol(n = 8) and 100 pmol (n = 7) VIP. ⁄P < 0.05 vs vehicle.

Table 1R–R interval, SBP values and BRS during the 15–25 min period after intra-arterialinjection of 0 (vehicle), 25, 50 and 100 pmol PACAP or VIP.

Peptide Doses(pmol)

n R–R interval(msec)

SBP (kPa) BRS (msec/kPa)

PACAP 0 (vehicle) 13 1302 ± 62 3.48 ± 0.11 2541 ± 30325 7 1291 ± 79 3.63 ± 0.16 2087 ± 24950 11 1330 ± 69 3.55 ± 0.13 2286 ± 344100 6 1297 ± 75 3.97 ± 0.26 1804 ± 279

VIP 0 (vehicle) 14 1369 ± 42 3.56 ± 0.09 2397 ± 13725 7 1330 ± 92 3.80 ± 0.15 2371 ± 42850 12 1442 ± 81 4.21 ± 0.21 2071 ± 219100 7 1127 ± 66 4.96 ± 0.16* 1434 ± 182*

n, number of trout.* P < 0.05 vs vehicle.

F. Lancien et al. / General and Comparative Endocrinology 171 (2011) 245–251 249

trout [46], as in mammals [33], are due to rhythmic sympatheticvasomotor activity. Following PACAP injection in trout, the overallSBP variability is enhanced, notably within the 0–0.1 Hz frequency

band, suggesting that central PACAP may enhance the vasomotorsympathetic activity. However, further studies using specificblockers of the autonomic nervous system may help to understandthe origin of SBP variability in trout and also the mechanism bywhich central PACAP enhances the power of the LF-SBP. After ICVinjection of vehicle or PACAP and within the 0.1–0.2 Hz frequencyrange of the PSD of the two signals, (1) the coherence between thespectra of the R–R and SBP variabilities is high (>0.5), and (2) anappropriate negative phase delay exists, indicating that within thisfrequency band, the input signal (the SBP) drives the output signal(the R–R interval). Consequently, we choose this frequency rangefor subsequent analysis of the transfer gain to give an estimate ofthe BRS. In vehicle-injected trout, this gain is about 2700 ms/Hz,a value similar to that previously calculated in trout using atime-domain method, the sequence technique [22]. ICV injectionof PACAP dose-dependently reduced the BRS. Although ICV injec-tion VIP had no significant effect on cardiovascular variables, its ac-tion on the spectral parameters of the R–R and SBP variabilitieswere quite similar to those of ICV PACAP. VIP also reduced theBRS with the same potency as PACAP. This result would indicatethat, within the brain of the trout, PACAP and VIP reduce the BRSby binding principally to VPAC receptors. The current data indicatethat the BRS is a more sensitive cardiovascular parameter than theR–R intervals or SBP to detect the central cardiovascular actions ofPACAP and VIP and perhaps those of other peptides.

A high dose of VIP injected within the periphery increased SBPwithout changing the R–R intervals and VIP reduced the BRS. In thecod Gadus morhua, VIP is also known to increase PDA withoutchanging the heart rate [17]. The mechanisms involved in the car-diovascular actions of VIP, particularly its effect on BRS, are un-known and several hypotheses can be proposed. Since VIP isthought to be an endogenous vasodilatatory neuropeptide in trout[18], the hypertensive effect observed following VIP injectionmight be indirect and arise, for example, from adrenaline actingon the vascular a-adrenoreceptors. In support of this idea, VIP ispresent in the vicinity of the chromaffin cells of the rainbow trout[35] and VIP causes the release of adrenaline from the in situ sal-ine-perfused trout posterior cardinal vein [30,31]. Concurrently,the absence of bradycardia and consequently the diminished BRSmight be attributable to a positive chronotropic action of the pep-tide on cardiac tissue. VIP-immunoreactivity is localised to thecholinergic postganglionic parasympathetic neurons of sino-atrialtissue of teleost [8]. Alternative explanations are that adrenalineacting via b-adrenergic receptors on the heart counteracts the bar-oreflex response or that high dose of VIP could diffuse to criticaltarget sites in the brain that lack the blood–brain barrier to reducethe BRS.

In teleost fish, the parasympathetic nervous system plays a cru-cial role in controlling the cardiac baroreflex, and consequently

250 F. Lancien et al. / General and Comparative Endocrinology 171 (2011) 245–251

cardiac output, through the action of the vagal nerve acting onmuscarinic cholinergic receptors in the heart [3,42,40,38,22].Therefore, we postulate that the change in the spontaneous cardiacBRS observed following ICV injection of PACAP and VIP may beexclusively mediated by the parasympathetic nervous system.The neural pathways involved in the central cardiovascular effectof PACAP and VIP cannot be determined from the present studyand further investigations will be needed to clarify this situation.Since PACAP and VIP were injected in close proximity to the preop-tic nucleus (NPO) they might mimic the action of the endogenouspeptides that are present within neuronal perikarya of this dience-phalic nucleus [27,29,45,26]. PACAP-, VIP- and also arginine vaso-tocin- and isotocin-containing preoptic neurons project not only tothe hypohysis but also towards the mesencephalon and the medul-la oblongata [29,26] where these neurohormonal peptides may af-fect the activity of cardiovascular nuclei including the nucleustractus solitarius and the dorsal vagal motor nucleus [4,36]. Finally,after ICV injection, PACAP and VIP may diffuse downstream withinthe cerebrospinal fluid towards critical cardiovascular nuclei in thebrainstem. It is worth mentioning that in the rat, intrathecal andICV injections of PACAP altered autonomic outflow and increasedheart rate but the effect on arterial blood pressure was less consis-tent [21,13,41]. In the rat, PACAP and PACAP receptors are alsopresent within the paraventricular nucleus, a nucleus homologousto the teleostean NPO [2,44]. PACAP excites paraventricular neu-rons [44], some of which have direct projection to autonomic neu-rons in the brainstem and spinal cord where they may contributeto the increase in sympathetic nerve activity and the depressionof BRS increasing the risk cardiovascular disease and hypertension[37,7].

The functional consequence of PACAP and VIP depression of thecardiac BRS in trout is unknown. It may be assumed that the spon-taneous cardiac baroreflex is responsible for the beat-to-beat phys-iological maintenance of resting blood pressure. Consequently,central PACAP and VIP, by causing a dose-dependent reduction inthe BRS, may thus affect blood pressure homeostasis leading tohigh blood pressure with possible consequences in terms of (1)cardiac performance, (2) mechanical damage to the delicate struc-ture of gill capillaries that might impinge on ions and water fluxesand gas exchange, and even (3) abnormal blood pressure responsesto internal or environmental stimuli.

In conclusion, our findings indicate new roles for PACAP andVIP, functioning as CNS neurotransmitter or neuromodulator pep-tides, for the control of neural pathways involved in the cardiacbaroreflex sensitivity in trout.

Acknowledgments

The authors thank Stéphanie Deshayes for her excellent techni-cal help during the course of this study and care in the mainte-nance of the animals. This work was supported by grants fromthe Institut National de la Santé et de la Recherche Médicale U650.

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