Central pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) decrease the baroreflex sensitivity in trout
Frédéric Lanciena, Nagi Mimassia, J. Michael Conlonb and Jean-Claude Le Mévela*
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, France.
b Department of Biochemistry, Faculty of Medicine and Health Sciences, United Arab Emirates University, 17666 Al Ain, United Arab Emirates.
Running title: Central PACAP and VIP on cardiac baroreflex
Number of text pages: 19
Number of figures: 4
Number of table: 1
* Author for correspondence (e-mail: email@example.com)
Although PACAP and VIP exert diverse actions on heart and blood vessels along the vertebrate phylum, no information is currently available concerning the potential role of these peptides on the regulation of the baroreflex response, a major mechanism for blood pressure homeostasis. Consequently, the goal of this study was to examine in our experimental model, the unanesthetized rainbow trout Oncorhynchus mykiss, 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 calculate the coherence, phase and gain of the transfer function between spontaneous fluctuations of systolic arterial blood pressure and R-R intervals of the electrocardiogram. The BRS was estimated as the mean of the gain of the transfer function when the coherence between the two signals was high and the phase negative. Compared with vehicle, intracerebroventricular (ICV) injections of trout PACAP-27 and trout VIP (25-100 pmol) dose-dependently reduced the cardiac BRS to the same extent with a threshold dose of 50 pmol for a significant effect. When injected intra-arterially at the same doses as for ICV injections, only the highest dose of VIP (100 pmol) significantly attenuated the BRS. These results suggest that the endogenous peptides PACAP and VIP might be implicated in the central control of cardiac baroreflex functions in teleosts.
Keywords: PACAP, VIP, R-R interval and systolic blood pressure variabilities, baroreflex, intracerebroventricular injection, intra-arterial injection, transfer function analysis, teleost.
Pituitary adenylate cyclase-activating polypeptide (PACAP) was originally isolated from ovine hypothalamus on the basis of its ability to stimulate adenylate cyclase activity in adenohypophysial cells (Miyata et al., 1989). PACAP is found in two biologically active forms, a 38 amino-acid peptide (PACAP-38) and a C-terminally truncated 27 amino-acid peptide (PACAP-27). In humans, the N-terminal portion of PACAP shows 68 % sequence identity with vasoactive intestinal peptide (VIP), identifying PACAP as a member of the VIP/secretin/glucagon/GH-releasing hormone superfamily of peptides (Sherwood et al., 2000). PACAP and VIP exert their biological activities through three G-protein coupled receptors termed PAC1, VPAC1 and VPAC2 (Laburthe et al., 2007). Consistent with the wide distribution of PACAP and VIP, and also their receptors, throughout the central nervous system and periphery, PACAP and VIP exert multiple actions (Vaudry et al., 2009). On the peripheral cardiovascular system, PACAP and VIP are considered to have potent and direct vasodilatory properties on a variety of arterial blood vessels and also to exert stimulatory effects on the heart (Farnham and Pilowsky, 2010; Vaudry et al., 2009). Nonetheless, the role of these peptides in central cardiovascular regulation is not so clearly delineated (Farnham and Pilowsky, 2010). In particular, nothing is known about the possible action of central PACAP and VIP on baroreflex, a key mechanism for blood pressure homeostasis.
The baroreflex has been evolutionary conserved from Agnatha (lamprey) to humans (Sundin and Nilsson, 2002; Karemaker and Wesseling, 2008). The baroreflex in fish, as in humans, is working spontaneously under baseline conditions (Lancien et al., 2007) and responds to adverse blood pressure changes (Sundin and Nilsson, 2002; Sandbloom and Axelsson, 2005). This baroreflex response is probably used as a mechanism to protect the delicate vasculature of the fish gills against high blood pressure (Bagshaw, 1985; Sundin and Nilsson, 2002). Additionally, the biologically active region of PACAP, (the N-terminal 27 amino acids), the sequence of VIP, and the primary structure of PACAP/VIP receptors have been remarkably well conserved from fish to humans (Wong et al., 1998; Sherwood et al., 2000; Montpetit et al., 2003). In teleost fish, the PACAP/VIP system is widely distributed in peripheral tissues (Reid et al., 1995; Kagstrom and Holmgren, 1997; de Girolamo et al., 1998) and in the central nervous system (Montero et al., 1998). We recently described the cardiorespiratory actions of these peptides after peripheral and central administration in trout (Le Mével et al., 2009). After intracerebroventricular (ICV) injection, PACAP and to lesser extend VIP provoked an hyperventilatory effect but only PACAP produced an increase in mean dorsal aortic blood pressure (PDA) without changing heart rate. Intra-arterial (IA) injections of either PACAP or VIP were without effect on ventilation and only VIP significantly elevated PDA without changing heart rate. The lack of heart rate response to elevation of blood pressure suggests that the cardiac baroreflex sensitivity (BRS) may be depressed following central PACAP and IA VIP. Therefore, the aim of this study was to examine in our experimental model, the unanesthetized rainbow trout Oncorhynchus mykiss, whether PACAP and VIP are involved in the regulation of the BRS. For this purpose, trout PACAP and VIP were injected within the third ventricle of the brain and intra-arterially and the modern transfer function analysis technique was used to study the cardiac BRS.
Materials and methods
Peptides and Chemicals
Trout PACAP-27 (HSDGIFTDSYSRYRKQMAVKKYLAAVL.NH2) and trout VIP (HSDAIFTDNYSRFRKQMAVKKYLNSVLT.NH2) were synthesized by GL Biochem (Shanghai, China) and purified to near homogeneity (> 98% purity) by reversed-phase HPLC. The identities of the peptides were confirmed by electrospray mass spectrometry. Peptides were stored in stock solution (0.01% HCl ) at –25 °C. For injections, the peptides were diluted to the desired concentration with Ringer’s solution (vehicle) immediately prior to use. The composition 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 µm filters (Millipore, Molsheim, France) before injection.
Animals and surgical procedures
Some of the results reported in the present paper refer to recordings made during our own previous study on the action of PACAP and VIP in trout (Le Mével et al., 2009). Recordings were excluded from the analysis if they contained excessive artefacts on electrocardiogram (ECG) signal or on pulsatile blood pressure. Additional new experiments were also carried out on rainbow trout Oncorhynchus mykiss using experimental procedures that have been described in detail in previous work (Le Mével et al., 2009).
Briefly, rainbow trout (body wt 240-270 g) were equipped with two electrocardiographic electrodes, a dorsal cannula, and an ICV microguide with a buccal cathether that was used to record the buccal ventilatory pressure (not quantified in the present study). After surgery, the animals were transferred to a 6 liter blackened chamber supplied with dechlorinated and aerated tap water (10-11 °C) that was both recirculating and through-flowing. Oxygen pressure within the water tank (PwO2) and pH were continuously recorded and maintained at constant levels (PwO2 = 20 kPa; pH = 7.4-7.6). The trout were allowed to recover from surgery and to become accustomed to their new environment for 48-72 h. Experimental protocols were approved by the Regional Ethics Committee in Animal Experiments of Brittany, France.
Intracerebroventricular and intra-arterial administrations of PACAP and VIP
For all protocols, the recording session lasted 30 min and all injections were made at the fifth minute of the test.
For the ICV protocol, the fish received an ICV injection of vehicle (0.5 µl) or an ICV injection of trout PACAP or VIP (25, 50 and 100 pmol in 0.5 µl).
For the IA protocol, 50 µl of vehicle, trout PACAP or VIP at doses of 25, 50, and 100 pmol was injected through the dorsal aorta cannula and immediately flushed by 150 µl of vehicle. Peptides were administered in random order.
Data acquisition and transfer function analysis of the cardiovascular variables
The ECG and PDA signals were recorded using standardized electronic devices (Le Mével et al., 2009). The output signals were digitalized at 1000 Hz, visualized on the screen of a PC during the 30-min recording period and 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 were processed off-line with custom-made programs written in LabView 6.1 (Laboratory Virtual Instrument Engineering Workbench, National Instruments, Austin, USA). For all protocols, 10 min segments of ECG and PDA signals were selected 15 min after ICV or IA injections of vehicle, PACAP, or VIP. R-R intervals were determined after detection of the R waves from the ECG recordings and systolic blood pressure (SBP) was identified from the pulsatile PDA. Their mean values were calculated. R-R interval and SBP time series were resampled at 2.56 Hz to obtain equidistant data points. The linear trend was removed from this new time series and 11 segments of 256 data points (100 sec) overlapping by half were subjected to a Hanning window.
Transfer function analysis
Spectral and cross spectral techniques developed in the present study were adapted from methods described previously in human (deBoer et al., 1985, Parati et al., 2000), rat (Japundzic et al., 1990), and lizard (De Vera and Gonzalez, 1997). The power spectral density (PSD) of each segment was calculated using standard fast Fourier transform and the PSD spectrum obtained were averaged. In order to investigate to what extent the input signal (the SBP) influences the output signal (the R-R interval) the coherence, phase and transfer function spectra of SBP against R-R interval were determined. The coherence spectrum, which has values between 0 and 1, is a measure of the correlation between the variations of the two signals. The transfer function provides a measure of the degree to which input signal content, at a given frequency, appears in output energy. A high coherence (>0.5) between the two signals and a negative phase shift indicates that the SBP mediates the changes in R-R intervals. Consequently, the cardiac BRS was estimated as the mean of the gain of the transfer function when the coherence was high and the phase negative.
All calculations for R-R interval (in msec), SBP (in kPa), PSD (in kPa²/Hz), coherence, phase function (in sec) and transfer gain (in kPa/msec) were made for the post-injection period of 20-30 min and the results were averaged for trout subjected to the same protocol.
Data are expressed as means S.E.M or +S.E.M. In the figures and table, data refer to absolute values . For comparison between groups, Kruskal-Wallis non-parametric one-way analysis of variance followed by Dunn’s multiple comparison test was used. A value of P < 0.05 was considered significant. The statistical tests were performed and the graphs constructed using GraphPad Prism 5.0 (GraphPad, San Diego, CA).
Baroreflex response to central PACAP
Fig. 1 shows a representative example of 5 min R-R interval time-series, and SBP time series recorded during the 20-30 min period in a trout receiving firstly an ICV injection of vehicle (Fig. 1A) followed 30 minutes later by an ICV injection of 50 pmol PACAP (Fig. 1B). Comparing the PACAP-injected trout with the vehicle-injected trout, PACAP did not produce any obvious change in the mean R-R interval value but decreased the R-R interval variability. Moreover, PACAP provoked an increase in SBP and following the injection, the SBP time-series showed more clearly shaped and enhanced low frequency oscillations. Fig. 2 shows an example of the average results obtained during the different steps of the transfer function analysis of 10 min R-R interval and SBP time-series for all trout receiving an ICV injection of vehicle or PACAP (50 pmol). For the PSD of the R-R interval variability, 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 0.1-0.2 Hz and exhibiting the highest PSD (Fig. 2A). For the PSD of the SBP variability, only a main LF component appeared in the 0-0.1 Hz frequency band peaking at 0.04 Hz (Fig. 2B). The ICV injection of PACAP decreased the PSD of the HF peak of the R-R interval variability (Fig. 2A) but amplified the LH peak (Fig. 2B). In contrast, after PACAP the PSD of the SBP-LF band increased (Fig. 2B). Following ICV injection of vehicle or PACAP a high coherence (>0.5) between the two signals was observed within the 0.1-0.2 Hz frequency range (Fig. 2C). The negative phase between the two signals in the 0.1-0.2 Hz frequency band after ICV vehicle or PACAP indicates that the input signal (the SBP) drives the output signal (the R-R interval) (Fig. 2D). The transfer gain, giving an estimate of the BRS, shows that the BRS in the 0.1-0.2 Hz frequency band is decreased following PACAP injection compared to vehicle -injected trout (Fig. 2E). The histograms in Fig. 3 summarize the average changes in R-R interval, SBP and in the transfer function gain between SBP and R-R interval spectra after ICV injection of vehicle or a range of doses (25-100 pmol) of PACAP. Compared with vehicle-injected trout, PACAP produced a dose-dependent increase in SBP but no change in R-R intervals (Fig. 3A). Fig. 3B demonstrates that the transfer function gain, i.e. the BRS, is dose-dependently decreased following ICV PACAP.
Baroreflex response to central VIP
The effect of VIP on the cardiovascular variables and cardiac BRS are summarized in Fig. 4. In contrast to PACAP, the ICV injection of VIP (25-100 pmol) elicited a non-significant increase in SBP and a non-significant decrease in R-R intervals (Fig. 4A). Nonetheless, ICV VIP produced a significant and dose-dependent decrease in the gain of the transfer function compared to vehicle-injected trout (Fig. 4B), a response that is identical to that observed following ICV PACAP (Fig. 3B). Consequently, the cardiac BRS is also decreased following central VIP.
Similar computerized transfer function analyses of the cardiovascular variables were performed after peripheral administration of vehicle, PACAP and VIP. After each treatment, the coherence between the PSD of the R-R interval and the SBP variabilities was high and the phase was negative within the 0.1-0.2 Hz frequency band (not shown). Table 1 summarizes all changes observed in the cardiovascular variables, and transfer function gain after the various treatments. Peripheral PACAP did not produce any significant change in the R-R interval and SBP values compared to vehicle injection and, in contrast to central PACAP, the BRS is not influenced by peripheral PACAP (Tab. 1). Table 1 also shows that only the highest dose of peripheral VIP (100 pmol) significantly increased the SBP and non- significantly reduced the R-R intervals compared to the peripheral administration of vehicle. Moreover, at this high dose, VIP significantly reduced the transfer function gain compared to vehicle injection (Table 1)
To our knowledge, our results demonstrate for the first time in any vertebrate class that exogenous administration of the two neurohormonal peptides PACAP and VIP in the brain reduces the cardiac BRS. The effects of the two peptides are most probably mediated primarily by the central nervous system since peripheral injection of VIP reduced the BRS only at its highest dose. The results have been obtained using the modern signal processing technique, transfer function analysis, that allows determination of the spontaneous cardiac baroreflex from cross-spectral analysis of the variabilities in R-R intervals of the ECG and SBP (deBoer et al., 1985; Japundzic et al., 1990; Parati et al., 2000). This technique obviates the need to use of intra-arterial administration of hypertensive and hypotensive pharmacological drugs to evoke reciprocal cardiac baroreflex responses. These substances may diffuse to the CNS and may produce confounding effects on the baroreflex responses . As well as providing an appropriate method to estimate the gain of the spontaneous cardiac baroreflex, transfer function analysis also offers during its various steps an attractive method to analyze the rhythmic oscillations spontaneously occurring in R-R intervals and SBP (Parati et al., 2000). This is exemplified in Fig. 2 of the present work and we will focus the discussion on the characteristics of the transfer function analysis after ICV injection of vehicle and PACAP since the action of VIP was virtually identical to that of PACAP.
The spectral analysis of R-R interval variability, also called the heart rate variability (HRV), after ICV injection of vehicle gave a result which is consistent with previous studies in trout (De Vera and Priede, 1991; Le Mével et al., 2002) and demonstrated that HRV had two fundamental components: a LF component within the 0-0.1 Hz frequency band and a HF component within the 0.1-0.2 Hz frequency band. Cardiac vagotomy or atropine administration abolished HRV in teleost fish (Altimiras et al., 1995; Le Mével et al., 2002; Campbell et al., 2004) while propanolol injection was without significant action on HRV (Altimiras et al., 1995) demonstrating that the vagal parasympathetic nervous system is the main, or even the exclusive, contributor to HRV in teleost fish (Grossman and Taylor, 2007). After ICV injection, PACAP decreased the R-R interval variability suggesting that PACAP reduced the cardiac vagal outflow from the brainstem. To the best of our knowledge, spectral analysis of SBP had never been described in fish. The LF component of the SBP variability which peaked between 0.03-0.04 Hz (25-33 sec period; Fig. 1B) suggests that the oscillations in the SBP might correspond to Mayer waves. Mayer waves have already been described in trout (Wood, 1974; Le Mével and Mabin, 1985). Mayer waves in humans have a characteristic frequency of about 0.1 Hz (van de Borne et al., 1997) and 0.4 Hz in rats (Brown et al., 1994). It is generally accepted that arterial pressure Mayer waves in trout (Wood, 1974), as in mammals (Pagani et al., 1997), are due to rhythmic sympathetic vasomotor activity. Following PACAP injection in trout , the overall SBP variability is enhanced, notably within the 0-0.1 Hz frequency band, suggesting that central PACAP may enhance the vasomotor sympathetic activity. However, further studies using specific blockers of the autonomic nervous system may help to understand the origin of SBP variability in trout and also the mechanism by which central PACAP enhances the power of the LF-SBP. After ICV injection of vehicle or PACAP and within the 0.1-0.2 Hz frequency range of the PSD of the two signals, (1) the coherence between the spectra of the R-R and SBP variabilities is high (> 0.5), and (2) an appropriate negative phase delay exists, indicating that within this frequency band, the input signal (the SBP) drives the output signal (the R-R interval). Consequently, we choose this frequency range for subsequent analysis of the transfer gain to give an estimate of the BRS. In vehicle-injected trout, this gain is about 2700 msec/Hz, a value similar to that previously calculated in trout using a time- domain method, the sequence technique (Lancien et al., 2007). ICV injection of PACAP dose-dependently reduced the BRS. Although ICV injection VIP had no significant effect on cardiovascular variables, its action on the spectral parameters of the R-R and SBP variabilities were quite similar to those of ICV PACAP. VIP also reduced the BRS with the same potency as PACAP. This result would indicate that, within the brain of the trout, PACAP and VIP reduce the BRS by binding principally to VPAC receptors. The current data indicate that the BRS is a more sensitive cardiovascular parameter than the R-R intervals or SBP to detect the central cardiovascular actions of PACAP and VIP and perhaps those of other peptides.
A high dose of VIP injected within the periphery increased SBP without changing the R-R intervals and VIP reduced the BRS. In the cod Gadus morhua, VIP is also known to increase PDA without changing the heart rate (Jensen et al., 1991). The mechanisms involved in the cardiovascular actions of VIP, particularly its effect on BRS, are unknown and several hypotheses can be proposed. Since VIP is thought to be an endogenous vasodilatatory neuropeptide in trout (Kagstrom and Holmgren, 1997), the hypertensive effect observed following VIP injection might be indirect and arises, for example, from adrenaline acting on the vascular -adrenoreceptors. In support of this idea, VIP is present in the vicinity of the chromaffin cells of the rainbow trout (Reid et al., 1995) and VIP causes the release of adrenaline from the in situ saline-perfused trout posterior cardinal vein (Montpetit and Perry, 1999; 2000). Concurrently, the absence of bradycardia and consequently the diminished BRS might be attributable to a positive chronotropic action of the peptide on cardiac tissue. VIP-immunoreactivity is localised to the cholinergic postganglionic parasympathetic neurons of sino-atrial tissue of teleost (Davies et al., 1994). Alternative explanations are that adrenaline acting via β-adrenergic receptors on the heart counteracts the baroreflex response or that high dose of VIP could diffuse to critical target sites in the brain that lack the blood - brain barrier to reduce the BRS.
In teleost fish, the parasympathetic nervous system plays a crucial role in controlling the cardiac baroreflex, and consequently cardiac output, through the action of the vagal nerve acting on muscarinic cholinergic receptors in the heart (Bagshaw, 1985; Taylor et al., 1999; Sundin and Nilsson, 2002; Sandbloom et al., 2005, Lancien et al., 2007). Therefore, we postulate that the change in the spontaneous cardiac BRS observed following ICV injection of PACAP and VIP may be exclusively mediated by the parasympathetic nervous system. The neural pathways involved in the central cardiovascular effect of PACAP and VIP cannot be determined from the present study and further investigations will be needed to clarify this situation. Since PACAP and VIP were injected in close proximity to the preoptic nucleus (NPO) they might mimic the action of the endogenous peptides that are present within neuronal perikarya of this diencephalic nucleus (Matsuda et al., 1997; Montero et al., 1998; Wong et al., 1998; Mathieu et al., 2001). PACAP- , VIP-, and also arginine vasotocin- and isotocin- containing preoptic neurons project not only to the hypohysis but also towards the mesencephalon (Montero et al., 1998; Mathieu et al., 2001) and the medulla oblongata (Montero et al., 1998; Mathieu et al., 2001) where these neurohormonal peptides may affect the activity of cardiovascular nuclei including the nucleus tractus solitarius and the dorsal vagal motor nucleus (Batten et al., 1990; Saito et al., 2004). Finally, after ICV injection, PACAP and VIP may diffuse downstream within the cerebrospinal fluid towards critical cardiovascular nuclei in the brainstem. It is worth mentioning that in the rat, intrathecal and ICV injections of PACAP altered autonomic outflow and increased heart rate but the effect on arterial blood pressure was less consistent (Lai et al., 1997; Farnham et al., 2008; Tanida et al., 2010). In the rat, PACAP and PACAP receptors are also present within the paraventricular nucleus, a nucleus homologous to the teleostean NPO (Arimura et al., 1991; Vaudry et al., 2009). PACAP excites paraventricular neurons (Vaudry et al., 2009) some of which have direct projection to autonomic neurons in the brainstem and spinal cord where they may contribute to the increase in sympathetic nerve activity and the depression of BRS increasing the risk cardiovascular disease and hypertension (Saleh, 2003; Dampney et al., 2005).
The functional consequence of PACAP and VIP depression of the cardiac BRS in trout is unknown. It may be assumed that the spontaneous cardiac baroreflex is responsible for the beat-to-beat physiological maintenance of resting blood pressure. Consequently, central PACAP and VIP, by causing a dose-dependent reduction in the BRS, may thus affect blood pressure homeostasis leading to high blood pressure with possible consequences in terms of (1) cardiac performance, (2) mechanical damage to the delicate structure of gill capillaries that might impinge on ions and water fluxes and gas exchange, and even (3) abnormal blood pressure responses to internal or environmental stimuli.
In conclusion, our findings indicate new roles for PACAP and VIP, functioning as CNS neurotransmitter or neuromodulator peptides, for the control of neural pathways involved in the cardiac baroreflex sensitivity in trout.
The authors thank Stéphanie Deshayes for her excellent technical help during the course of this study and care in the maintenance of the animals. This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale U650.
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Legends to figures
Fig. 1. Example illustrating 5 min R-R interval and SBP beat- to- beat time-series, 15 min after intracerebroventricular injection of (A) 0.5 l vehicle, and (B) 50 pmol PACAP in the same unanesthetized trout. Note that following PACAP, the mean R-R interval did not obviously change but the variability in R-R intervals decreased, while the SBP value increased and low frequency oscillations appeared more clearly in the SBP time-series.
Fig. 2. Average power spectral density (PSD) for (A) R-R interval variability and (B) SBP variability, (C) coherence, (D) phase and (E) transfer gain between the two signals after intracerebroventricular injection of vehicle (continuous line, n=9) and 50 pmol PACAP (hatched line, n=9). LF : low frequency; HF: high frequency. For statistical analysis see Fig. 3.
Fig. 3 Histograms showing (A) the R-R intervals (scale on the left) and the SBP values (scale on the right), (B) the BRS during the 15-25 min period after intracerebroventricular injection of 0 (vehicle, n=11), 25 pmol (n=8), 50 pmol (n=9) and 100 pmol (n= 10) PACAP. * P < 0.05 vs vehicle.
Fig. 4 Histograms showing (A) the R-R intervals (scale on the left) and the SBP values (scale on the right), (B) the BRS during the 15-25 min period after intracerebroventricular 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 1. R-R interval, SBP values and BRS during the 15-25 min period after intra-arterial injection of 0 (vehicle), 25 pmol, 50 pmol and 100 pmol PACAP or VIP. n, number of trout, * P < 0.05 vs vehicle.