PACAP release from the canine adrenal gland in vivo: functional role in adrenomedullary response to severe hypotension

This study was to investigate if endogenous pituitary adenylate cyclase-activating polypeptide (PACAP) can be released during direct splanchnic nerve stimulation in vivo, and to determine whether PACAP in the adrenal gland can modulate the medullary response to sympathoadrenal reflex. The output of adrenal catecholamine and PACAP38-like immunoreactivity (PACAP-38-ir) increased in a frequency-dependent manner following direct splanchnic nerve stimulation (0.2–20 Hz). The both responses were highly reproducible and PACAP-38-ir output closely correlated with catecholamine output. Sodium nitroprusside (SNP; 0.1 mg/kg, iv, bolus) caused a severe hypotension resulting in marked increases in catecholamine secretion. In the presence of local PACAP-27 (125 ng), the maximum catecholamine response to SNP was significantly potentiated in a synergistic manner as compared with that obtained in the group receiving SNP or PACAP-27 alone. The study indicates that endogenous PACAP-38 can be released particularly when the sympathoadrenal system is highly activated and that the local exogenous PACAP-27 enhanced the reflex-induced catecholamine release, suggesting collectively a facilitating role of PACAP as neuromodulator in the sympathoadrenal function in vivo. Role of PACAP In Sympathoadrenal Regulation 3 Introduction The adrenal medullae release catecholamines in response to various stresses that activates the sympathetic nervous system. It is well established that acetylcholine (ACh), the classical cholinergic neurotransmitter released from splanchnic nerve terminals, evokes catecholamine secretion (7). In addition to ACh, it has been acknowledged that various endogenous neuropeptides are co-released along with ACh to mediate and/or modulate catecholamine secretion in response to varying stress. Among several neuropeptides playing a potential role as a non-cholinergic neurotransmitter and/or neuromodulator in the adrenal medulla, pituitary adenylate cyclase-activating polypeptide (PACAP) is likely to play a major role. PACAP is a neuropeptide having two isoforms composed of 27 and 38 amino acids (PACAP-27 and PACAP-38), which are both widely distributed in the central and peripheral nervous systems. The rat adrenal gland contains high concentrations of PACAP as well as PAC1 receptors (29). PACAP immunoreactive nerve fibers ending on chromaffin cells have also been identified in the rat adrenal gland (6, 12, 27). A few studies in vitro have shown that endogenous PACAP was released in response to direct splanchnic nerve stimulation in isolated, perfused porcine and rat adrenal glands (27, 30) and that the direct nerve stimulation-induced catecholamine secretion was diminished by a PACAP antagonist (8). These previous observations in vitro have suggested that PACAP may play a role of controlling adrenomedullary function during an actual stimulation of the sympathoadrenal system in vivo. However, such a functional implication of PACAP in the sympathoadrenal system in physiological and/or Role of PACAP In Sympathoadrenal Regulation 4 pathophysiological situations still remains mostly conjectural in the limited number of experimental demonstrations under in vivo conditions. Therefore, the present study was carried out: (i) to determine whether endogenous PACAP can be actually released into the adrenal venous effluent during a direct splanchnic nerve stimulation in vivo, and (ii) to investigate if locally administered PACAP can effectively modulate the adrenal catecholamine secretion in response to a reflex activation of the sympathoadrenal system in anesthetized dogs. Methods Surgical preparations of the animals. Adult mongrel dogs fasted overnight but allowed free accesses to water were anesthetized with pentobarbital sodium (30 mg/kg iv, followed by 4 mg/kg as needed). Artificial respiration was maintained through an endotracheal tube with a Harvard pump (model 607). The rectal temperature was monitored and kept constant at 37.5 ± 0.5°C by means of a thermoregulator (model 74, Yellow Springs Instruments, Yellow Springs, OH) connected to a heating pad. Physiological saline was slowly administered intravenously during the whole period of the experiment to prevent dehydration. The pH of the physiological saline was adjusted at 7.38 immediately before use. Both femoral arteries were cannulated: the right femoral artery was used to measure aortic pressure and the left femoral artery to obtain aortic blood samples. Preparation for direct splanchnic nerve stimulation. The direct splanchnic nerve stimulation was applied in the group of animals in which endogenous PACAP-38 was Role of PACAP In Sympathoadrenal Regulation 5 determined in the left adrenal venous effluent. After a median laparotomy and a left flank incision, the left splanchnic nerve was dissected free from surrounding tissues, double ligated ~ 2 cm from the adrenal gland, and protected from dryness by applying mineral oil. Direct electrical stimulation was applied to the distal end of this nerve. All the other nerves to the left adrenal gland from the lumbar paravertebral sympathetic ganglia and from the celiac-superior mesenteric plexus were double ligated and cut within ~ 2 cm from the gland to prevent undesired retrograde nerve conduction during direct splanchnic nerve stimulation (9, 35). Preparation of local intra-arterial drug infusion to the left adrenal gland. Following a median laparotomy and a left flank incision, the left adrenolumbar artery was dissected free from surrounding tissue for installation of a cannula in a retrograde manner, i.e., from the peripheral end towards the gland and aorta (34). The catheter (PE90) was inserted so that the tip reached beneath the gland or to a point close to the adrenolumbar arterial-aortic junction. All visible branches arising from the adrenolumbar artery toward the outside of the gland were ligated to prevent undesired drug diffusion into the systemic circulation. The volume of this catheter was fixed at 0.5 ml, and the catheter was connected to an infusion pump (model 1140-001, Harvard). PACAP-27 was locally infused to the left adrenal gland through this catheter at a fixed rate of 0.5 ml/min. Preparation of the extracorporeal adrenal venous circuit. Adrenal venous blood was sampled through a polyethylene catheter (PE-240) specially shaped at one end for easy introduction and inserted into the left adrenolumbar vein via the left femoral vein. Role of PACAP In Sympathoadrenal Regulation 6 The catheter was tied at the adrenoabdominal-vena cava junction to prevent adrenal venous blood contamination with abdominal vena cava blood. The left adrenolumbar vein, distal to the gland, was ligated to obtain actual adrenal venous blood. Venous blood from the gland was drained into a small blood reservoir filled with saline. The volume of this catheter was fixed at 1.5 ml. Blood volume in the reservoir was kept as small as possible and the venous blood was returned to the dog using an automatic blood level controller connected to an infusion pump (Masterflex model 7016-52, Cole-Parmer Instrument, Chicago, IL) through a catheter inserted into the right femoral vein at a perfusion rate adjusted to a stabilized initial venous flow (34). Preparation of acute surgical denervation of the left adrenal gland. In a separate group, a relative contribution of the local splanchnic input to the adrenal medulla was tested during a reflex activation of the sympathoadrenal system induced by a severe hypotension with an intravenous injection of sodium nitroprusside (SNP; Sigma Chemical, St-Louis, MO). To this end, an acute surgical denervation of the left adrenal gland was performed by cutting the left splanchnic nerve according to our previously reported procedure (35). All the other nerves to the left adrenal gland where cut following the same method described for preparing the direct splanchnic nerve stimulation. After all surgical procedures were completed, sodium heparin (200 U/kg iv) was administered, followed by 100 U/kg every hour thereafter. Dogs were then allowed a stabilization period of ~ 60 min. Role of PACAP In Sympathoadrenal Regulation 7 Measured parameters. Mean aortic pressure and heart rate were measured and recorded with a polygraph system (model RM-6000, Nihon-Kohden, Tokyo, Japan). Aortic and left adrenal venous blood was simultaneously sampled into graded, chilled tubes for catecholamine analyses. In experiments with direct splanchnic nerve stimulation, plasma concentrations of endogenous PACAP-38 were also determined in the same blood samples served for catecholamine measurements. Adrenal venous blood flow was determined by a gravimetric method at each sampling time point (34). Hematocrit was measured in all adrenal venous blood samples. For the catecholamine measurements, an aliquot of 1.5 ml of blood was transferred to a centrifuge tube containing 30 μl of preservative solution (pH 6.5) consisting of ethylene glycol-bis (βamino-ethyl ether)-N,N,N’,N’-tetraacetic acid (95 mg/ml) and glutathione (60 mg/ml). These blood samples were immediately centrifuged at 4 °C for 5 min at 14,000 revolutions/min with a refrigerated centrifuge (model 5402, Eppendorf, Hamburg, Germany). Plasma was stored at –80 °C until assayed. Plasma concentrations of epinephrine and norepinephrine were quantified by means of an isocratic high performance liquid chromatographic system (Gilson, Villiers-Le-Bel, France) coupled with an electrochemical detector “Coulochem II” (model 5200; ESA, Bedford, MA) (34). At the end of each experiment, the left adrenal gland was removed and weighed. Adrenal catecholamine data were obtained in net catecholamine output calculated as follows: net output of adrenal catecholamine (ng·min·g of gland) = ([CA]AV -[CA]AO) × BFAV × (1HctAV)/wet weight of gland, where [CA]AV is plasma catecholamine concentration in adrenal venous blood, [CA]AO is plasma catecholamine concentration in aortic blood, BFAV is adrenal venous blood flow, and HctAV is adrenal venous blood hematocrit. For Role of PACAP In Sympathoadrenal Regulation 8 aortic and adrenal venous PACAP-38 measurements, an aliquot of 4 ml of blood was transferred to a chilled polypropylene test tube containing disodium EDTA (1 mg/ml of blood). Plasma PACAP-38 concentration was determined by radioimmunoassay with a commercial kit (RIK-8920, Peninsula Laboratories, San Carlos, CA). The protease inhibitor aprotinin was not used in this study, because we have previously shown that peptide concentrations in plasma were similar either in the presence or absence of aprotinin at the concentration of 500 KIU/ml of blood (10). Experimental protocol. The present study consisted of three separate series of experiments. The first series contained one experimental group (29.9 ± 1.2 kg; n = 6) that served to determine if endogenous PACAP-38 is actually released into the adrenal venous effluent during a direct splanchnic nerve stimulation under in vivo conditions. In this group, the distal end of tightly double-ligated left splanchnic nerve was stimulated with bipolar platinum electrodes by rectangular pulses of supramaximal voltage (12 V) by means of an electronic stimulator (model SEN-3301; Nihon Kohden). The stimulation period consisted of three successive stimuli with three different frequencies, 0.2, 2, and 20 Hz, each lasting 2 min, without interruption between each stimulus. The pulse duration was also modified in such a manner that the splanchnic nerves received the identical total pulse duration during each stimulus, which corresponds to a single pulse duration of 20, 2, and 0.2 ms for each frequency of 0.2, 2, and 20 Hz, respectively (10). Adrenal venous blood was separately collected into a chilled, graded tube during each stimulus (2 min), for a total period of 6 min. The dead volume of the adrenal venous catheter (1.5 ml) was taken into account for blood collection during each stimulus. Aortic Role of PACAP In Sympathoadrenal Regulation 9 blood samples (4 ml) were simultaneously obtained during adrenal venous sample collections at every sampling time point. After a 30 min stabilization period, another control sample of aortic and adrenal venous blood was taken, and a second stimulation period was applied under exactly the same conditions to ensure the reproducibility of the peptide response. The second series involved four groups of experiments. This series served to evaluate whether the presence of locally administered PACAP-27 (Sigma Chemical, StLouis, MO) could modulate the adrenal catecholamine secretion in response to a reflex activation of the sympathoadrenal system during the SNP-induced severe hypotension. The experiments of this series were designed so that a possible interaction between SNP (depicting the reflex-mediated splanchnic neural drive to the adrenal medulla) and PACAP-27 can be evaluated by means of a two-way analysis of variance. The first group (21.5 ± 1.2 kg, n = 8) received a local infusion of the vehicle (saline 0.9%, pH 7.38) combined with a simultaneous intravenous bolus injection of the vehicle and served as the control. One minute after the initial control sample was taken, the vehicle was infused into the left adrenolumbar artery at a rate of 0.5 ml/min for 5 min. The dead volume of the adrenal arterial catheter (0.5 ml) was taken into account in relation to the infusion rate. Similarly, the dead volume of the adrenal venous catheter (1.5 ml) was taken into consideration in relation to adrenal venous blood flow while adrenal venous blood samples were collected. At the onset of vehicle infusion (designated min 0), a bolus injection of the vehicle was given intravenously. An adrenal venous blood Role of PACAP In Sympathoadrenal Regulation 10 sample was obtained during the first min of vehicle infusion (0-1 min) with a delay depending on the venous blood flow, followed by sample collections at 1-2, 2-3, 3-4, and 4-5 min during the whole period of vehicle infusion lasted for 5 min. Samples were also collected at 5-6, 6-7, 10-11, 15-16, 30-31 and 45-46 min after cessation of the vehicle infusion. The duration of each adrenal venous sample collection was thus fixed to 1 min. Aortic blood samples (1.5 ml each) were simultaneously obtained during adrenal venous sample collection at these sampling points. The second group (29.2 ± 3.5 kg, n = 8) received a local infusion of the vehicle combined with a simultaneous intravenous bolus injection of SNP (0.1 mg/kg; Sigma Chemical) and served as the SNP-induced hypotension group. The experimental protocol for this group followed the same procedures as those described above. The third group (23.5 ± 1.6 kg, n = 8) received a total dose of 125 ng of PACAP27 given locally to the left adrenal gland via the left adrenolumbar artery with the concentration of 50 ng/ml at the infusion rate of 0.5 ml/min for precisely 5 min. Instead of SNP, a bolus of saline was given intravenously at the onset of PACAP-27 infusion. This group served as the PACAP-induced catecholamine secretion group. The procedures for drug administrations and sample collections were exactly the same as those described for the vehicle control group. After taking an aliquot of 1.5 ml of adrenal venous blood (for plasma catecholamine quantification) from the first seven samples obtained during and after the drug infusion, the remaining blood was not return to the dog to prevent potential systemic hemodynamic changes due to the released catecholamine. Role of PACAP In Sympathoadrenal Regulation 11 Finally, the fourth group (26.8 ± 3.4 kg, n = 8) received a local infusion of PACAP-27 (125 ng as a total dose, see above) to the left adrenal gland combined with a bolus intravenous injection of SNP (0.1 mg/kg) at the onset of PACAP-27 infusion. The protocol for blood sampling followed exactly the same procedures described for the vehicle control group. The third series, consisted of one group (26.9 ± 1.6 kg, n = 8), served to assess the relative contribution of the splanchnic neural pathway in the sympathoadrenal counterresponse to SNP-induced severe hypotension. This group received both the acute surgical denervation of the left adrenal gland and intravenous bolus injection of SNP (0.1 mg/kg, iv bolus). The vehicle was locally infused to the left adrenal gland. The experimental protocols for the sequence of the drug administration as well as the timing for sample collections of adrenal venous and aortic blood were exactly the same as those described for the second series of experiments. The concentration of PACAP-27 used in this study was selected on the basis of our previous observations obtained under similar experimental conditions (36). The period of PACAP-27 infusion, however, was fixed for 5 min to ensure the presence of PACAP-27 in the adrenal gland during a period of the maximal sympathoadrenal reflex stimulation, which could be estimated to be about 5 min according to the time-course of adrenal catecholamine secretion following the intravenous administration of SNP alone. The dose of SNP was chosen on the basis of several dose-finding pilot experiments in Role of PACAP In Sympathoadrenal Regulation 12 which a bolus intravenous injection of SNP caused a severe fall in arterial pressure from which the animals could recover over a given experimental period. Each of the experimental protocols described for all series was applied only once in each animal. All surgeries and experimental procedures were carried out while the dogs were under full surgical anesthesia. All experiments were acute, terminal procedures. The experimental protocols have been approved by the animal research committee of the Université de Montréal. The animals used in this study have been cared for and used in accordance with the Guiding principles for research involving animals and human beings published by the American Physiological Society (ref. Am J Physiol Regul Integr Comp Physiol 283: R281-R283, 2002). Statistical analyses. The statistical evaluation was made using a package of statistical software (SigmaStat for Windows, version 2.03; SPSS, Chicago, IL). Differences over a given experimental period were assessed by an analysis of variance for repeated measures followed by multiple comparisons with one control using the Dunnett’s method. A two-way analysis of variance was conducted to assess possible interaction between PACAP-27 and SNP. This test was made on the maximal net amount of catecholamines released during the first 5 min following the SNP administration and/or PACAP-27 infusion. When applicable, a preliminary logarithmic transformation was used to satisfy the condition of a normal distribution of variance (32). All results are expressed as means ± SE, and P < 0.05 was considered statistically significant. Role of PACAP In Sympathoadrenal Regulation 13 Results Responses to a direct splanchnic nerve stimulation. Values for plasma concentrations of catecholamines and PACAP-38-ir in adrenal venous and aortic blood, mean arterial pressure, heart rate and adrenal venous blood flow obtained before and during splanchnic nerve stimulation are summarized in Table 1. During the first stimulation period, both adrenal venous catecholamine concentrations and blood flow raised in response to the three successive stimuli following a frequency-dependent manner, reaching a statistically significant level at the frequencies of 2 and 20 Hz. PACAP-38-ir concentrations also increased in a frequency-dependent manner and reached a significant level at the highest frequency tested (20 Hz), at which the concentration of PACAP-38-ir was approximately two-fold higher than the basal value (Table 1). Aortic blood catecholamine and PACAP-38-ir concentrations and hemodynamic parameters remained unchanged. Consequently, the output of adrenal catecholamines and PACAP-38-ir increased in a similar profile to that observed in their concentrations (Fig. 1). The catecholamine and PACAP-38-ir responses to the direct splanchnic nerve stimulation returned to their respective basal control levels by 30 min after the cessation of the first bloc of stimulation. These secretory responses were highly reproducible upon repetition of the subsequent stimulation given with an interval of 30 min (Fig. 1). Furthermore, the correlation analyses revealed that the release of PACAP38-ir strongly correlated with epinephrine (r = 0.915, n = 8, P = 0.0002) and norepinephrine (r = 0.909, n = 8, P = 0.0002) outputs. Role of PACAP In Sympathoadrenal Regulation 14 Responses to a severe hypotension. In the group receiving SNP alone, the administration of SNP caused an immediate and marked decrease (~ 60%) in the mean arterial pressure (Fig. 2C). During this hypotensive period, the plasma catecholamine concentrations (Table 2) and outputs (Fig. 2A-B) increased markedly, reaching the maximum level at least ~ 15 (norepinephrine concentration) to ~ 25 (epinephrine concentration) times higher than the corresponding control value observed before the SNP administration. The onset of catecholamine responses to the fall in arterial pressure was rapid, and the increased catecholamine outputs returned to their corresponding control levels ~ 15 min following the SNP administration (Fig. 2A-B). Plasma catecholamine concentrations in aortic blood and heart rate also increased in response to the severe hypotension (Table 2) while adrenal venous blood flow decreased (Table 2). Responses to a local PACAP-27 infusion. In the group receiving PACAP-27 alone, the intra-arterial infusion of PACAP-27 to the left adrenal gland with a dose of 125 ng, did not cause significant changes in circulating catecholamine concentrations in aortic blood, mean arterial pressure, and heart rate. Adrenal venous catecholamine concentrations and blood flow increased significantly (Table 2). Consequently, the output of both epinephrine and norepinephrine increased in response to PACAP-27 infusion (Fig. 2A & B). This increase in catecholamine output was rapid, and the peak response was observed during the infusion. The increased catecholamine output returned to the corresponding control levels ~10 min after the cessation of infusion. Role of PACAP In Sympathoadrenal Regulation 15 Catecholamine responses to PACAP-27 during severe hypotension. An important fall in mean arterial pressure occurred immediately after the bolus intravenous injection of SNP following a similar profile to that obtained in dogs receiving SNP alone. The adrenal catecholamine output increased markedly in response to severe hypotension with a concomitant local infusion of PACAP-27 (Fig. 2A & B). Aortic catecholamine concentrations and heart rate also increased while, adrenal venous blood flow did not change significantly (Table 2). The changes in the net catecholamine responses, defined as delta (∆) catecholamine output, observed during the infusion of the vehicle alone, the SNP-induced hypotension alone, the PACAP-27 alone, and the SNP-induced hypotension combined with the local PACAP-27 infusion are summarized in Fig. 3. Those catecholamine responses were calculated from the peak responses obtained during the first 5 min following SNP injection and/or PACAP-27 infusion. In the presence of PACAP-27, the maximum increase in catecholamine secretion during SNP-induced hypotension was significantly greater than that observed in the group receiving either SNP or PACAP27 alone (Fig. 3). Furthermore, a two-way analysis of variance performed on these maximum responses revealed the presence of a significant interaction between the SNPand the PACAP-mediated catecholamine responses (P = 0.005 for epinephrine; P = 0.039 for norepinephrine), indicating a synergistic interaction between these two factors. Responses to severe hypotension in dogs with left adrenal denervation. In the group receiving the acute left adrenal denervation, a bolus intravenous injection of SNP Role of PACAP In Sympathoadrenal Regulation 16 caused an immediate and marked decrease in the mean arterial pressure (Fig. 4C). However, both outputs and plasma concentrations of epinephrine and norepinephrine increased only slightly in response to the severe hypotension (Fig. 4A & B, Table 3). The maximum net catecholamine outputs during SNP-induced hypotension were significantly attenuated by ~ 85% (P = 0.045 for epinephrine; P = 0.021 for norepinephrine) in the group receiving the acute left adrenal denervation as compared to those obtained in dogs with the normal left adrenal innervation (Fig. 5A & B). Aortic epinephrine and norepinephrine concentrations, and heart rate also increased in response to severe hypotension (Table 3). Adrenal venous blood flow decreased significantly during the SNP challenge (Table 3). Discussion The aims of the present study were to evaluate if endogenous PACAP could be actually released into the adrenal venous effluent during direct splanchnic nerve stimulation in vivo; and to determine whether the presence of exogenous PACAP in the adrenal gland could locally modulate the adrenomedullary function during sympathoadrenal reflex activation in anesthetized dogs. Direct splanchnic nerve stimulation caused significant increases in the release of both adrenal catecholamines and PACAP-38-ir in a frequency-dependent manner, showing strong correlations between the output of catecholamines and PACAP-38-ir. The results also indicate that the adrenal catecholamine response to the SNP-induced severe hypotension was significantly enhanced in a synergistic manner in the presence of PACAP-27 administered locally to the adrenal gland, showing a facilitating interaction between these two factors. Role of PACAP In Sympathoadrenal Regulation 17 In the present study, we measured endogenous PACAP-38-ir in the local adrenal venous blood under basal conditions as well as during direct splanchnic nerve stimulation, since the tissue distribution of endogenous PACAP-38 predominates over the other isomer (2), and because immunoreactive PACAP has been localized to splanchnic nerve fibers innervating chromaffin cells in the adrenal medulla (6, 12, 26, 27). Our present data show for the first time in vivo that endogenous PACAP-38 could be released into the adrenal venous effluent during direct splanchnic nerve stimulation following a frequencydependent manner and reaching a statistical significance with the highest frequency (20 Hz) tested. This in vivo finding is consistent with previous in vitro observations in that the release of endogenous PACAP-38 during either transmural or direct nerve stimulation occurred only when the relatively high frequencies (10 – 16 Hz) were applied in the isolated, perfused rat (30) and porcine (27) adrenal gland. In addition, we also found in this study that the stimulation-induced release of PACAP-38-ir could be closely correlated with that of epinephrine and norepinephrine under in vivo conditions. Although the adrenal catecholamine secretory response is primarily controlled by the classical neurotransmitter, acetylcholine (12), the present observation may imply the existence of functional interaction in the adrenal gland between endogenous PACAP and catecholamine secretions by modulating the cholinergic neurotransmission during the sympathoadrenal activation in certain physiopathological situations (12, 15). Under certain conditions associated with stress such as severe hypotension, a reflex activation of the sympathoadrenal system occurs to rapidly restore the cardiovascular homeostasis (16). In the present experiment, the intravenous injection of Role of PACAP In Sympathoadrenal Regulation 18 SNP caused a rapid and severe fall in mean arterial blood pressure associated with an immediate and significant rise in the adrenal catecholamine secretion. It has been well documented that the hypotensive effects of intravenous SNP are due to peripheral vasodilatation and reduction in peripheral resistance as a result of the action of released nitric oxide on the vascular beds (1, 28). The immediate adrenomedullary response to the SNP-induced hypotension, therefore, may have resulted from the baroreflex-mediated sympathoadrenal stimulation, since the left adrenal catecholamine response was diminished by about 85% in dogs receiving the acute surgical denervation of the left adrenal gland. With respect to the validity of the acute surgical denervation used in the present study, we have previously shown that the centrally mediated adrenomedullary response to bilateral carotid occlusion was almost completely diminished in the denervated left adrenal gland (35). In the present study, plasma catecholamine concentrations in aortic blood significantly increased during the SNP-induced hypotension in dogs with the denervated left adrenal gland. This increase in circulating catecholamines may be due most probably to the increased catecholamine output from the right adrenal gland that has been kept normally innervated. Thus, the lack of the catecholamine response in the left denervated adrenal gland during the severe hypotension ensures the functional existence of the splanchnic neural pathway mediating the medullary secretion during the reflex activation of the sympathoadrenal system. While the two PACAP isoforms are very potent secretagogues in the adrenal medulla both in vitro and in vivo (29), we have previously shown that under the similar experimental conditions, the amplitude of adrenal catecholamine response to PACAP-27 Role of PACAP In Sympathoadrenal Regulation 19 was at least three times greater than that obtained with PACAP-38 on the same molar basis in anesthetized dogs (11). In the present experiments, we therefore used PACAP-27 to ensure the adrenal catecholamine response to PACAP. In dogs receiving PACAP-27 alone, the local administration of this peptide to the normally innervated adrenal gland resulted in marked increases in the catecholamine secretions without significantly affecting the mean arterial blood pressure. The catecholamine response to PACAP-27 may have resulted most probably from its specific action on the PAC1 receptor, as the presence of PACAP6-27, a selective PAC1 receptor antagonist (22, 23), in the adrenal gland almost completely blocked the PACAP-27-induced catecholamine response under similar experimental conditions in anesthetized dogs (18). Even if PACAP6-27 may have certain affinity for VPAC2 receptor as in the case of PACAP6-38 (13), this possibility does not seem to have direct relevance to the adrenal gland in which the PAC1 receptor is almost exclusively expressed (19). Moreover, the observation that arterial blood pressure did not significantly change during PACAP-27 infusion is consistent with the view that the PACAP-27-induced increase in catecholamine secretion resulted from its local action on the adrenal medulla and not from secondary reflexive effects due to hypotension that could also be produced by PACAP when administered intravenously (24, 25). Adrenal venous blood flow remained statistically unchanged during the concomitant administration of PACAP-27 and SNP, while increased with PACAP-27 alone and decreased with SNP alone. It has been demonstrated that in anesthetized dogs, SNP-induced hypotension with a decrease of mean arterial pressure of ~ 55% caused a significant reduction of blood flow in the ascending aorta, celiac, superior mesenteric, Role of PACAP In Sympathoadrenal Regulation 20 renal and iliac arteries (3). In the light of these observations, the severe hypotension (~ 60% of control) provoked in the group receiving SNP alone might have induced a similar reduction of blood flow in the abdominal region, ultimately resulting in a diminished blood supply to the adrenal gland. On the other hand, PACAP is also known to be a potent vasodilator (24, 25). In the present study, however, PACAP-27 was locally administered to the adrenal gland without producing any significant systemic effects. Since PACAP is such a potent vasodilator, the elevation in adrenal blood flow during a single infusion of PACAP-27 alone may have resulted from its local vasodilating effect. Therefore, when both SNP and PACAP-27 were administered concomitantly through their respective routes (intravenously and local arterial infusion, respectively), it is most likely that PACAP-27 evoked a local adrenal vasodilatation that counteracted a decrease in adrenal blood supply due to the SNP-induced severe hypotension. The net result of both effects is finally illustrated by a null gain in the adrenal venous blood flow. The synergistic effect of PACAP-27 on the adrenal catecholamine secretion, observed in the present study, may have resulted most probably from the local interaction of PACAP-27 with the operative splanchnic neurotransmission largely mediated by neural acetylcholine in the adrenal medulla during the SNP-induced hypotension. In this context, we have previously shown that in the similar experimental setup, locally applied PACAP-27 synergistically enhanced the adrenal catecholamine secretion induced by either direct splanchnic nerve stimulation or the local administration of acetylcholine to the adrenal gland in anesthetized dogs (17). Taken together, these previous and the present observations suggest that the presence of PACAP in the adrenal medulla Role of PACAP In Sympathoadrenal Regulation 21 enhances adrenal catecholamine secretion either by facilitating the release of neural acetylcholine or by postsynaptic multiple intracellular interactions with various second messengers (31). While a potential PACAP-induced presynaptic facilitation of acetylcholine release has been suggested by the enhanced cardiac parasympathetic cholinergic neurotransmission in anaesthetized dogs (14, 25), the site of interaction in the canine adrenal gland cannot precisely be determined in the present study. With respect to a potentially functional role of either endogenous PACAP-27 or PACAP-38, both peptides would theoretically play a similar role of facilitating the catecholamine secretion, because both peptides have the same binding affinity to PAC1 receptor (13). The relative potency, however, as judged from the catecholamine releasing effects of exogenous PACAP27 and PACAP38 on the canine adrenal gland (11), must be greater with PACAP27 if the latter peptide can be actually released along with PACAP38. Nevertheless, it has been reported that, in the porcine adrenal gland in vitro, the tissue content of PACAP-27 was found to be only 1% of that of PACAP-38, and that, perhaps more importantly, PACAP-27 could not be detected in the adrenal venous perfusate following direct splanchnic nerve stimulation (27). As endogenous PACAP38 could be released into the venous effluent in the porcine adrenal in vitro (27) as well as in the canine adrenal in vivo during direct splanchnic nerve stimulation as observed in this study, it is conceivable that PACAP-38 rather than PACAP-27 may play a role of a neuromodulator to facilitate the sympathoadrenal activity in a certain physiopathological situation. Nevertheless, a possible implication of endogenous PACAP-27 in the canine adrenal gland in vivo cannot completely be excluded in the present study. Role of PACAP In Sympathoadrenal Regulation 22 Under the present experimental conditions, however, the magnitude of adrenal catecholamine release during the SNP-induced hypotension is roughly equivalent to or even smaller than that obtained at 2 Hz in the group receiving direct splanchnic nerve stimulation. Based on this rather over-simplified comparison, it appears unlikely that a significant amount of endogenous PACAP can be released from splanchnic nerve endings during severe hypotension. On the other hand, however, it must be noted that the concentration of endogenous PACAP-38 found in this study was obtained in the adrenal venous effluent, leaving a possibility that its intrasynaptic concentration at the adrenomedullary synapse may be much more elevated to an extent sufficient to modulate the cholinergic neurotransmission. Also, it has been well documented that the general anesthesia including the one with pentobarbital-Na considerably blunts peripheral responses to the baroreceptor-mediated reflexes (5). Under a less suppressive anesthesia, the SNP-induced hypotension may have generated a greater sympathoadrenal response, a condition under which endogenous PACAP may be preferentially released from the splanchnic nerves. In conclusion, the present study demonstrates that endogenous PACAP-38 can be released in the adrenal medulla during direct splanchnic nerve stimulation at a preferably high frequency under in vivo conditions. The study also indicates that the presence of exogenous PACAP-27 in the adrenal gland potentiates in a synergistic manner the catecholamine secretion during SNP-induced severe hypotension. Taken together, these results are compatible with the view that PACAP may play a functional role as a local Role of PACAP In Sympathoadrenal Regulation 23 neuromodulator in facilitating or sustaining adrenal catecholamine secretion during severe activation of the sympathoadrenal system. However, the actual pathophysiological implication of endogenous PACAP-38 remains to be further investigated under various reflex-induced sympathoadrenal stimulations. Perspectives In the present study, we used SNP as a pharmacological tool to induce a rapid and severe hypotension that provoked a strong baroreflex-mediated activation of the sympathoadrenal system. As the SNP-induced hypotension results from a decrease in peripheral resistance due to the vasodilating effect of nitric oxide released from the SNP molecule (1), it is conceivable that the released nitric oxide may affect the multiple mechanisms controlling the adrenal catecholamine secretion. In this context, it has been shown that in the isolated, perfused canine adrenal gland, endogenous nitric oxide inhibited the nicotine-induced catecholamine release (4). In contrast, however, nitric oxide produced a large stimulation of the basal catecholamine secretion as well as the evoked catecholamine release induced by low concentrations of nicotine in cultured bovine chromaffin cells (21). Therefore, in parallel to the present study, we have tested direct in vivo effect on the adrenal gland of SNP locally administered in the same way as in the case of PACAP-27 infusion. Within the dose range of SNP (0.065 μg – 6.5 μg per adrenal gland), there was no significant direct effect on the basal adrenal catecholamine secretion in anesthetized dogs. With the highest dose tested (6.5 μg per adrenal gland), however, the basal catecholamine secretion increased slightly due to the indirect, baroreflex-induced activation of the sympathoadrenal system. Thus, nitric oxide released Role of PACAP In Sympathoadrenal Regulation 24 from the SNP molecule does not directly affect the basal catecholamine secretion in the canine adrenal gland under in vivo conditions. This observation is consistent with a previous study that, in the canine adrenal gland in vivo, another nitric oxide donor, NOC 7, had no direct effect on the basal adrenal catecholamine secretion (20). Nevertheless, either the potentially facilitating or inhibiting effect of SNP-derived nitric oxide on the PACAP-27-induced adrenal catecholamine secretion remains to be determined under conditions where the operative sympathoadrenal system is absent to define whether the observed synergism results from the interaction either with the neural factors or with the SNP-derived nitric oxide. Role of PACAP In Sympathoadrenal Regulation 25 Acknowledgements The authors wish to thank Sanae Yamaguchi for her skilled and devoted technical assistance. The authors are also grateful to Jean-Marc Chianetta and Eric Himaya, supported by the Merck-Frosst Summer Research Program for 2000 and 2001, respectively, for their active participation to the present study. This work was supported by the grants from the Canadian Institutes of Health Research (MT-10605). Stéphane Lamouche is a holder of a doctoral research award from the Canadian Institutes of Health Research. Role of PACAP In Sympathoadrenal Regulation26 References1. Al-sa’doni H, and Ferro A. S-Nitrosothiols: a class of nitric oxide-donor drugs.Clin Sci (Lond) 98: 507 – 520, 2000. 2. Arimura A, Somogyvari VA, Miyata A, Mizuno K, Coy DH, and Kitada C.Tissue distribution of PACAP as determined by RIA: highly abundant in the ratbrain and testes. Endocrinology 129: 2787-2789, 1991. 3. Bagshaw RJ, Cox RH, and Campbell KB. Sodium nitroprusside and regionalarterial haemodynamics in the dog. Br J Anaesth 49(8): 735-743, 1977. 4. Barnes RD, Ward LE, Frank KP, Tyce GM, Hunter LW, and Rorie DK.Nitric oxide modulates evoked catecholamine release from canine adrenalmedulla. Neuroscience 104: 1165-1173, 2001. 5. Cox RH, and Bagshaw RJ. Influence of anesthesia on the response to carotidhypotension in dogs. Am J Physiol Heart Circ Physiol 237: H424-H432, 1979. 6. Dun NJ, Tang H, Dun SL, Huang R, Dun EC, and Wakade AR. Pituitaryadenylate cyclase activating polypeptide-immunoreactive sensory neuronsinnervate rat adrenal medulla. Brain Res 716: 11-21, 1995. Role of PACAP In Sympathoadrenal Regulation27 7. Feldberg W, Minz B, and Tsudzimura H. The mechanism of nervous dischargeof adrenaline. J Physiol (Lond) 81, 286-304, 1934. 8. Fukushima Y, Hikichi H, Mizukami K, Nagayama T, Yoshida M, Suzuki-Kusaba M, Hisa H, Kimura T, and Satoh S. Role of endogenous PACAP incatecholamine secretion from the rat adrenal gland. Am J Physiol RegulatoryIntegrative Comp Physiol 281: R1562-R1567, 2001. 9. Gaspo R, Yamaguchi N, and de Champlain J. Effects of nifedipine and BAY K8644 on stimulation-induced adrenal catecholamine secretion in the dog. Am JPhysiol Regulatory Integrative Comp Physiol 265: R28-R34, 1993. 10. Gaspo R, Yamaguchi N, and de Champlain J. Correlation between neuralrelease of VIP and adrenomedullary catecholamine secretion in vivo. Am JPhysiol Regulatory Integrative Comp Physiol 268: R1449-R1455, 1995. 11. Geng G, Gaspo R, Trabelsi F, and Yamaguchi N. Role of L-type Ca channelin PACAP-induced adrenal catecholamine release in vivo. Am J PhysiolRegulatory Integrative Comp Physiol 273: R1339-R1345, 1997. 12. Hamelink C, Tjurmina O, Damadzic R, Young WS, Weihe E, Lee HW, andEiden LE. Pituitary adenylate cyclase-activating polypeptide is a Role of PACAP In Sympathoadrenal Regulation28 sympathoadrenal neurotransmitter involved in catecholamine regulation andglucohomeostasis. Proc Natl Acad Sci USA 99(1); 461-466, 2002. 13. Harmar AJ, Arimura A, Gozes I, Journot T, Laburthe M, Pisegna JR,Rawlings SR, Robberecht P, Said SI, Sreedharan SP, Wank SA, andWaschek JA. International Union of Pharmacology. Nomenclature of receptorsfor vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activatingpolypeptide (PACAP). Pharmacol Rev 50: 265–270, 1998. 14. Hirose M, Furukawa Y, Nagashima Y, Yamazaki K, Hoyano Y, and Chiba S.Effects of PACAP-38 on the SA nodal pacemaker activity in autonomicallydecentralized hearts of anesthetized dogs. J Cardiovasc Pharmacol 29: 216-221,1997. 15. Hökfelt T, Broberger C, David Xu Z-Q, Sergeyev V, Ubink R, and Diez M.Neuropeptides – an overview. Neuropharmacol 39: 1337-1356, 2000. 16. Ito K, Sato A, Shimamura K, and Swenson RS. Reflex changes in sympatho-adrenal medullary functions in response to baroreceptor stimulation inanesthetized rats. J Auton Nerv Syst 10: 295-303, 1984. Role of PACAP In Sympathoadrenal Regulation29 17. Lamouche S, Martineau D, and Yamaguchi N. Modulation of adrenalcatecholamine release by pituitary adenylate cyclase-activating polypeptide invivo. Am J Physiol Regulatory Integrative Comp Physiol 276: R162-R170, 1999. 18. Lamouche S, and Yamaguchi N. Role of PAC1 receptor in adrenalcatecholamine secretion induced by PACAP and VIP in vivo. Am J PhysiolRegulatory Integrative Comp Physiol 280: R510-R518, 2001. 19. Moller K, and Sundler F. Expression of pituitary adenylate cyclase activatingpeptide (PACAP) and PACAP type I receptors in the rat adrenal medulla. RegulPept 63: 129-139, 1996. 20. Nagayama T, Hosokawa A, Yoshida M, Suzuki-Kusaba M, Hisa H, KimuraT, and Satoh S. Role of nitric oxide in adrenal catecholamine secretion inanesthetized dogs. Am J Physiol Regulatory Integrative Comp Physiol 275:R1075-R1081, 1998. 21. Oset GM, Parramon M, Hortelano S, Bosca L, and Gonzalez M. Nitric oxideimplication in the control of neurosecretion by chromaffin cells. J Neurochem 63:1693-1700, 1994. 22. Robberecht P, Gourlet P, De Neef P, Woussen-Colle MC, Vandermeers-PiretMC, Vandermeers A, and Chrisstophe J. Structural requirements for the Role of PACAP In Sympathoadrenal Regulation30 occupancy of pituitary adenylate-cyclase-activating-peptide (PACAP) receptorsand adenylate cyclase activation in human neuroblastoma NB-OK-1 cellmembranes. Eur J Biochem 207: 239–246, 1992. 23. Robberecht P, Woussen-Colle M-C, De Neef P, Gourlet P, Buscail L,Vandermeers A, Vandermeers-Piret M-C, and Christophe J. The two formsof the pituitary adenylate cyclase activating polypeptide (PACAP(1-27) andPACAP(1-38)) interact with distinct receptors on rat pancreatic AR 4-2J cellmembranes. FEBS Lett 86: 133–136, 1991. 24. Runcie MJ, Ulman LG, and Potter EK. Effects of pituitary adenylate cyclase-activating polypeptide on cardiovascular and respiratory responses in anesthetizeddogs. Regul Pept 60: 193-200, 1995. 25. Seki Y, Suzuki Y, Baskaya MF, Saito K, Takayasu M, Shibuya M, and SugitaK. Central cardiovascular effects induced by intracisternal PACAP in dogs. Am JPhysiol Heart Circ Physiol 38: H135-H139, 1995. 26. Tabarin A, Chen D, Hakanson R, and Sundler F. Pituitary adenylate cyclase-activating polypeptide in the adrenal gland of mammals: distribution,characterization and responses to drugs. Neuroendocrinol 59: 113-119, 1994. Role of PACAP In Sympathoadrenal Regulation31 27. Tornoe K, Hannibal J, Jensen TB, Georg B, Rickelt LF, Andreasen MB,Fahrenkrug J, and Holst JJ. PACAP-(1-38) as a neurotransmitter in the porcineadrenal glands. Am J Physiol Endocrinol Metab 279: E1413-E1425, 2000. 28. Tuzel IH. Sodium nitroprusside: A review of its clinical effectiveness as ahypotensive agent. J Clin Pharmacol 14: 494 – 503, 1974. 29. Vaudry D, Gonzalez BJ, Basille M, Yon L, Fournier A, and Vaudry H.Pituitary adenylate cyclase-activating polypeptide and its receptors: from structureto functions. Pharmacol Rev 52: 269-324, 2000. 30. Wakade AR, Gou X, Strong R, Arimura A, and Haycock J. Pituitaryadenylate cyclase-activating polypeptide (PACAP) as a neurotransmitter in ratadrenal medulla. Regul Pept 37: 327, 1992. (Abstract) 31. Wakade AR. Multiple transmitter control of catecholamine secretion in ratadrenal medulla. Advances in Pharmacology 45: 595-598, 1998. 32. Wallenstein S, Zucker CL, and Fleiss J. Some statistical methods useful incirculation research. Circ Res 47: 1-9, 1980. 33. Watanabe T, Masuo Y, Matsumoto H, Suzuki H, Ohtaki T, Masuda Y,Kitada C, Tsuda M, and Fujino M. Pituitary adenylate cyclase-activating Role of PACAP In Sympathoadrenal Regulation32 polypeptide provoques cultured rat chromaffin cells to secrete adrenaline.Biochem Biophys Res Commun 182 (1): 403-411, 1992. 34. Yamaguchi N. In vivo evidence for adrenal catecholamines release mediated bynonnicotinic mechanism: local medullary effect of VIP. Am J Physiol RegulatoryIntegrative Comp Physiol 265: R766-R771, 1993. 35. Yamaguchi N, Lamarche L, and Briand R. Simultaneous evaluation ofmedullary secretory functions of normal and acutely denervated adrenals. Am JPhysiol Regulatory Integrative Comp Physiol 260: R306-R313, 1991. 36. Yamaguchi N and Lamouche S. Enhanced reactivity of the adrenal medulla inresponse to pituitary adenylate cyclase-activating polypeptide1-27 (PACAP) duringinsulin-induced hypoglycemia in anesthetized dogs. Can J Physiol Pharmacol 77:819-826, 1999. Role of PACAP In Sympathoadrenal Regulation33 Figure legendsFig. 1. Adrenal epinephrine (EPI, A), norepinephrine (NE, B) and PACAP-38-ir (C)output in response to splanchnic nerve stimulation at 0.2, 2, and 20 Hz. Two sets of stimulation were given at an interval of 30 min. C, control value observed immediatelybefore each stimulation period. * P < 0.05 compared with corresponding initial controlvalue. Fig. 2. Adrenal epinephrine (EPI, A) and norepinephrine (NE, B) output and mean arterialpressure (MAP, C) in the group receiving the vehicle (inversed open triangle; n = 8); asingle intravenous bolus dose of SNP (filled circle; n = 8); a local infusion of PACAP-27into the left adrenolumbar artery (partially filled triangle; n = 8); and a local infusion ofPACAP-27 combined with a bolus injection of SNP (partially filled square; n = 8).Arrow, injection of SNP. Horizontal bar, duration of PACAP-27 infusion. *P < 0.05compared with corresponding initial values for each group (open symbols). Fig.3. Maximum net increases (∆) in epinephrine (EPI) and norepinephrine (NE) output obtained during the first 5 min following SNP administration and/or PACAP-27 infusion.Maximum net responses in each group were calculated with the data obtained from thesecond series of experiments. A significant interaction exists between PACAP-27 andSNP, * P < 0.05, two-way analysis of variance. Fig. 4. Adrenal epinephrine (EPI, A) and norepinephrine (NE, B) output and mean arterialpressure (MAP, C) in response to a single bolus dose of SNP given intravenously in the Role of PACAP In Sympathoadrenal Regulation34 groups with either normal innervated (IG; partially filled square; n = 8) or acutedenervated left adrenal gland (SNX; filled circle; n = 8). Arrow, injection of SNP. *P <0.05 compared with corresponding initial values for each group (open symbols). Fig.5. Maximum net increases (∆) in epinephrine (EPI) and norepinephrine (NE) output obtained during the first 5 min following SNP administration. Maximum net response inthe group with either the normally innervated (IG) or acutely denervated (SNX) leftadrenal gland was calculated with the data obtained from the second and third series ofexperiments, respectively. *P < 0.05 between IG and SNX. Role of PACAP In Sympathoadrenal Regulation35 Table 1. Changes in plasma PACAP-38-ir and catecholamine concentrations and hemodynamic parameters before and during direct splanchnicnerve stimulation in dogs.Values are means ± SE obtained from 6 dogs. Samples were taken before (basal) and during splanchnic nerve stimulation at 0.2, 2 and 20 Hz. S1 and S2, 1 and 2 stim on given immediately after corresponding control; stimulation periods S1 and S2 were given with an interval of 30 min.PAC38AV, adrenal venous PACAP-38-ir (pg/ml);PAC38AO, aortic PACAP-38-ir (pg/ml); EPIAV, adrenal venous epinephrine (ng/ml); NEAV, adrenal venous norepinephrine (ng/ml); EPIAO, aortic epinephrine (ng/ml); NEAO, aorticnorepinephrine (ng/ml); MAP, mean arterial pressure (mmHg); HR, heart rate (beats/min); BFAV, adrenal venous blood flow (ml/2min). *P < 0.05 compared with correspondingcontrol values.ulatiS1S2Basal0.2 Hz2.0 Hz20 HzBasal0.2 Hz2.0 Hz20 HzPAC38AV 10.2 ± 1.2 12.1 ± 1.6 15.0 ± 2.1 19.6 ± 2.7* 10.7 ± 2.3 11.5 ± 3.2 18.0 ± 4.6 20.4 ± 2.2*PAC38AO 7.7 ± 1.1 8.4 ± 2.3 7.5 ± 0.8 5.9 ± 1.4 7.0 ± 1.0 7.6 ± 0.7 8.5 ± 1.37.8 ± 0.4EPIAV 21.2 ± 8.6 36.0 ± 9.0 312.9 ± 101.2* 823.6 ± 297.2* 18.7 ± 6.1 51.4 ± 11.8 499.6 ± 210.3* 1105.5 ± 298.8*NEAV 3.4 ± 1.5 4.7 ± 1.7 60.1± 17.4* 200.5 ± 61.0* 2.9 ± 1.4 6.7 ± 1.7 87.5 ± 35.4* 260.4 ± 70.2*EPIAO 0.08 ± 0.02 0.07 ± 0.02 0.07 ± 0.02 0.09 ± 0.02 0.07 ± 0.03 0.08 ± 0.02 0.09 ± 0.01 0.09 ± 0.02NEAO 0.16 ± 0.02 0.20 ± 0.05 0.23 ± 0.03 0.24 ± 0.03 0.17 ± 0.02 0.15 ± 0.02 0.19 ± 0.02 0.22 ± 0.03MAP 137.6 ± 5.5 139.3 ± 5.8 142.3 ± 6.8 141.1 ± 5.2 138.0 ± 5.8 139.8 ± 6.3 141.9 ± 6.7 143.9 ± 6.2HR 156.3 ± 4.6 159.8 ± 5.2 159.7 ± 5.1 156.8 ± 4.8 158.8 ± 4.8 158.8 ± 4.8 158.2 ± 4.9 157.2 ± 5.0BFAV 8.1 ± 1.1 8.3 ± 1.0 8.9 ± 1.0* 10.1 ± 1.1* 7.3 ± 1.1 7.4 ± 1.1 8.2 ± 1.1* 9.4 ± 1.2* Role of PACAP In Sympathoadrenal Regulation36 Table 2. Changes in plasma catecholamine concentrations and hemodynamic parameters in dogs receiving either SNP or PACAP-27 aloneor a combination of both treatments.Gr 1: Vehicle + Vehicle(n = 8)Gr 2: Vehicle + SNP(n = 8)Gr 3: PACAP-27 + Vehicle(n = 8)Gr 4: PACAP-27 + SNP(n = 8) BasalPeak change(0-5 min) BasalPeak change(0-5 min) BasalPeak change(0-5 min) BasalPeak change(0-5 min)EPIAV 12.1 ± 3.9 9.7 ± 3.5 7.1 ± 3.0 175.6 ± 81.4* 16.0 ± 9.3 437.9 ± 81.5* 26.7 ± 12.6 1185.2 ± 478.3*NEAV 1.9 ± 0.9 2.0 ± 0.9 1.2 ± 0.5 20.8 ± 10.2* 1.9 ± 0.7 36.4 ± 7.8* 5.11 ± 2.9 106.4 ± 38.7*EPIAO 0.09 ± 0.06 0.12 ± 0.03 0.04 ± 0.03 0.54 ± 0.20* 0.09 ± 0.04 0.13 ± 0.03 0.07 ± 0.06 0.66 ± 0.24*NEAO 0.20 ± 0.02 0.23 ± 0.3 0.19 ± 0.03 0.55 ± 0.10* 0.25 ± 0.04 0.13 ± 0.03 0.23 ± 0.05 0.58 ± 0.14*MAP 131.2 ± 12.0 126.2 ± 10.8 128.6 ± 8.0 41.0 ± 4.4* 129.2 ± 12.5 127.4 ± 13.2 126.8 ± 9.4 50.0 ± 6.8*HR 167.7 ± 7.3 169.3 ± 7.6 164.8 ± 5.5 194.7 ± 4.7 176.8 ± 7.0 178.7 ± 6.4 149.0 ± 5.7 184.0 ± 12.5*BFAV 3.4 ± 0.5 3.9 ± 0.4 3.9 ± 0.4 2.6 ± 0.4* 3.8 ± 0.4 4.9 ± 0.4* 4.1 ± 0.5 5.0 ± 1.1 Values are means ± SE. Gr, group; n, number of dogs in each group; SNP, sodium nitroprusside;EPIAV, adrenal venous epinephrine (ng/ml); NEAV, adrenal venousnorepinephrine (ng/ml); EPIAO, aortic epinephrine (ng/ml); NEAO, aortic norepinephrine (ng/ml); MAP, mean arterial pressure (mmHg); HR, heart rate (beats/min); BFAV,adrenal venous blood flow (ml/min). *P < 0.05 compared with corresponding control values. Role of PACAP In Sympathoadrenal Regulation37 Table 3. Changes in plasma catecholamine concentrationsand hemodynamic parameters in dogs with left adrenaldenervation receiving SNP.SNX + Vehicle + SNP(n = 8) BasalPeak change(0-5 min)EPIAV10.52 ± 4.9134.14 ± 12.57*NEAV1.57 ± 0.545.47 ± 2.06*EPIAO0.07 ± 0.020.57 ± 0.19*NEAO0.14 ± 0.290.44 ± 0.08*MAP137.8 ± 6.544.8 ± 4.4*HR164.0 ± 7.7195.1 ± 12.0*BFAV2.9 ± 0.21.5 ± 0.6* Values are means ± SE. SNX, left adrenal denervation; SNP, sodiumnitroprusside; n, number of dogs;EPIAV, adrenal venous epinephrine(ng/ml); NEAV, adrenal venous norepinephrine (ng/ml); EPIAO, aorticepinephrine (ng/ml); NEAO, aortic norepinephrine (ng/ml); MAP, meanarterial pressure (mmHg); HR, heart rate (beats/min); BFAV, adrenalvenous blood flow (ml/min). *P < 0.05 compared with correspondingcontrol values. Role of PACAP In Sympathoadrenal Regulation38 Role of PACAP In Sympathoadrenal Regulation39 Role of PACAP In Sympathoadrenal Regulation40 Role of PACAP In Sympathoadrenal Regulation41 Role of PACAP In Sympathoadrenal Regulation42