- Email: [email protected]
Activation of cholecystokinin (CCK1) and serotonin (5-HT3) receptors increases the discharge of pancreatic vagal afferents
European Journal of Pharmacology 601 (2008) 198–206
Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / e j p h a r
Endocrine Pharmacology
Activation of cholecystokinin (CCK1) and serotonin (5-HT3) receptors increases the discharge of pancreatic vagal afferents Bashair M. Mussa, Daniela M. Sartor, Anthony J.M. Verberne ⁎ University of Melbourne, Department of Medicine, Clinical Pharmacology and Therapeutics Unit, Austin Health, Heidelberg 3084, Victoria, Australia
a r t i c l e
i n f o
Article history: Received 24 October 2008 Accepted 3 November 2008 Available online 9 November 2008 Keywords: Pancreatic vagal afferent Cholecystokinin CCK1 receptor Serotonin 5-HT3 receptor
a b s t r a c t Cholecystokinin and serotonin are released from the gastrointestinal tract in response to the products of digestion and play critical roles in mediating pancreatic secretion via vago-vagal reflex pathways. This study was designed to investigate the effects of activation of cholecystokinin CCK1 and serotonin (5-hydroxytryptamine, 5-HT) 5-HT3 receptors on pancreatic vagal afferent discharge and to determine whether there is an interaction between these receptors. Male Sprague Dawley rats anaesthetised with isoflurane (1.5%/100% O2) were used in all experiments. The effects of systemic administration of cholecystokinin and the serotonin 5-HT3 receptor agonist phenylbiguanide on pancreatic vagal afferent discharge were recorded before and after administration of cholecystokinin CCK1 and serotonin 5-HT3 receptor antagonists. Cholecystokinin (0.1–10 µg/kg, i.v.) and phenylbiguanide (1 and 10 µg/kg, i.v.) increased pancreatic vagal afferent discharge dose-dependently. Cholecystokinin CCK1 receptor antagonists, lorglumide (10 mg/kg, i.v.) and devazepide (0.5 mg/kg, i.v.), reduced cholecystokinin- and phenylbiguanide-induced increases in pancreatic vagal afferent discharge significantly (n = 5, P b 0.05). On the other hand, serotonin 5-HT3 receptor blockade with granisetron (1 mg/kg, i.v.) or MDL72222 ([(1S,5R)-8-methyl-8-azabicyclo[3.2.1]octan-3-yl] 3,5-dichlorobenzoate; 0.1 mg/kg, i.v.) inhibited the pancreatic vagal afferent discharge responses to phenylbiguanide but not those to cholecystokinin. This study has confirmed that cholecystokinin and phenylbiguanide activate pancreatic vagal afferent discharge via activation of cholecystokinin CCK1 and serotonin 5-HT3 receptors, respectively. In addition, it has demonstrated that (i) the serotonin 5-HT3 agonist phenylbiguanide acts partly via an interaction with cholecystokinin CCK1 receptors, and (ii) the actions of cholecystokinin are not dependent on serotonin 5-HT3 receptor activation. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Classically, the control of pancreatic secretion has been viewed as being dependent on two separate mechanisms: (i) the release of gastrointestinal hormones into the circulation and (ii) a vago-vagal reflex pathway. However, a recent notion has suggested that these two mechanisms act synergistically and proposes that activation of a vagovagal reflex by gastrointestinal hormones such as cholecystokinin and serotonin (5-hydroxytryptamine, 5-HT) mediates a considerable portion of pancreatic secretion (Li et al., 2000; Li and Owyang, 1994, 1996; Li et al., 2001b). The vago-vagal reflex pathway that regulates pancreatic secretion consists of three major components: (i) duodenal and pancreatic vagal afferents which originate from pseudo-bipolar neurons in the nodose ganglion and project in a bidirectional fashion to the nucleus of the solitary tract in the dorsal medulla and peripherally to the duodenum and pancreas, (ii) visceroceptive neurons in the nucleus of solitary tract that integrate the vagal afferent input, and (iii) pancreatic preganglionic
⁎ Corresponding author. Tel.: +61 3 94965978; fax: +61 3 94593510. E-mail address: [email protected] (A.J.M. Verberne). 0014-2999/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2008.11.007
vagal neurons which project mainly from the dorsal vagal nucleus. More detail regarding the role of these pathways in control of pancreatic secretion can be found in recent reviews (Love et al., 2006; NiebergallRoth and Singer, 2001). The vago-vagal reflex model suggests that gastrointestinal hormones such as cholecystokinin and 5-HT activate receptors on vagal afferents which terminate onto and activate neurons within the nucleus of the solitary tact via glutamate receptors (Liao et al., 2005). These neurons, in turn, influence pancreatic preganglionic neurons projecting from the dorsal vagal nucleus and modulate the vagal output to the pancreas via glutamatergic and GABAergic inputs (Love et al., 2006; Mussa and Verberne, 2008; Travagli et al., 1991, 2006). It is of interest to further explore the actions of cholecystokinin and 5-HT on pancreatic vagal afferents since this represents an integral part of the vago-vagal reflex that contributes to control of pancreatic secretion. While several gastrointestinal hormones are known to contribute to regulation of pancreatic secretion (Chang and Chey, 2001; Chey and Chang, 2001), cholecystokinin and 5-HT are believed to play major roles (Li et al., 2001a, 2000, 2001b; Owyang, 1996; Owyang and Logsdon, 2004). Several lines of evidence support the view that cholecystokinin and 5-HT act in a paracrine fashion on duodenal vagal
B.M. Mussa et al. / European Journal of Pharmacology 601 (2008) 198–206
afferents to influence pancreatic secretion in humans, rats and dogs (Konturek et al., 1979; Li and Owyang, 1993, 1996; Li et al., 2001b; Soudah et al., 1992). Additionally, secretin is one of the principal hormones that regulates secretion of bicarbonate through a direct action on pancreatic acini and perhaps also via activation of vagal afferent pathways (Li et al., 1998, 2005). In vitro and in vivo studies have confirmed that cholecystokinin receptors present on duodenal vagal afferents are predominantly the cholecystokinin CCK1 receptor subtype (Moran et al., 1987; Moriarty et al.,1997; Zarbin et al., 1981). There is electrophysiological evidence for high and low affinity states of the cholecystokinin CCK1 receptor, although the high-affinity receptor appears to be more important for pancreatic secretion (Li et al., 1997, 1999). Duodenal vagal afferents express a high density of serotonin 5-HT3 receptors and these are distributed widely throughout the gastrointestinal tract (Li et al., 2001a; Raybould et al., 2003). Cholecystokinin and 5-HT are potent stimulators of cholecystokinin CCK1 and serotonin 5-HT3 receptors on vagal afferents, respectively, and activate vago-vagal reflexes that are involved in regulation of several digestive processes including pancreatic secretion (Li et al., 2001a, 1997, 2000). Vagal afferents convey sensory information from various visceral organs including the duodenum and the pancreas. Thus, it is likely that vagal afferents arising from the latter may also be involved in regulation of pancreatic secretion. We hypothesised that cholecystokinin and 5-HT may activate the pancreatic vagal afferents by acting on cholecystokinin CCK1 and serotonin 5-HT3 receptors on these afferents, respectively. Although an earlier study by Niijma had shown that intracarotid injection of cholecystokinin and 5-HT produced an increase in pancreatic vagal afferent discharge, the receptors involved were not identified (Niijima, 1981). In addition, several studies have identified an interaction between cholecystokinin CCK1 and serotonin 5-HT3 receptors (Aja, 2006; Hayes et al., 2004; Li et al., 2004; Saita and Verberne, 2003). In these studies, serotonin 5-HT3 receptors were shown to mediate part of the inhibitory actions of cholecystokinin on food intake or on the discharge of medullary presympathetic vasomotor neurons. In view of these findings, the current study was designed to (i) investigate the effects of systemic administration of cholecystokinin and the serotonin 5-HT3 receptor agonist phenylbiguanide on pancreatic vagal afferent discharge specifically, (ii) to confirm that these effects are mediated by cholecystokinin CCK1 and serotonin 5HT3 receptors, respectively, and (iii) to determine whether there is an interaction between these receptors. We demonstrated that cholecystokinin and phenylbiguanide activate pancreatic vagal afferent discharge via cholecystokinin CCK1 and serotonin 5-HT3 receptors, respectively, and that there is an interaction between these receptors at the level of the pancreatic vagal afferents. 2. Methods and materials 2.1. Animal preparations All animal experimental protocols were conducted according to the guidelines of the Ethical Review Committee of the Austin and Repatriation Medical Centre (Heidelberg, Victoria, Australia) and the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Experiments were performed on 40 male Sprague Dawley rats weighing between 250–350 g obtained from the Animal Resource Centre (Perth, Western Australia). Rats were housed in a temperaturecontrolled environment with a 12 hour light-dark cycle and given free access to water and chow. They were initially anaesthetised using isoflurane vapour and were then tracheostomized to allow artificial ventilation with 100% O2 (1 ml/100 g body weight, 50–60 breaths/ min) containing 1.5–1.7% isoflurane. The body temperature was maintained at 36–38 °C throughout the experiment using a servocontrolled heat pad and the adequacy of anaesthesia was assessed by
199
absence of a withdrawal reflex to paw pinch as well as the absence of an eye blink response to gentle probing of the cornea. The adequacy of anaesthesia was re-assessed every 15 min and the anaesthetic vapour concentration was increased when necessary. The left carotid artery and left jugular vein were cannulated to monitor the arterial blood pressure and heart rate and to allow intravenous administration of drugs, respectively. 2.2. Isolation of the cervical and pancreatic vagus A small incision was made along the right side of the neck to expose the right cervical vagus nerve which was dissected free from the sheath and connective tissues. The intact nerve was placed onto a pair of silver wire hook electrodes and covered with liquid paraffin and then embedded in silicone sealant (Kwik-Cast, World Precision Instruments, Sarasota, FL, USA). Effectiveness of the cervical vagal stimulation was confirmed by observation of an immediate bradycardia and depressor response following application of electrical stimulation (5 Hz, 0.5 ms pulse width, 500 µA, 20s) to the vagus. A midline incision was made through the abdomen to gain access to the pancreas. The spleen was retracted caudally and laterally and the stomach was retracted contralaterally to expose the splenic segment of the pancreas. The pancreatic vagus nerve was traced along the coeliac branch of the posterior subdiaphragmatic vagal trunk which enters the pancreas tissue via the splenic artery. It was dissected carefully at about 0.5 cm from the point at which the splenic artery itself branches off from the coeliac artery. The pancreatic vagus nerve was isolated from the surrounding connective tissue and placed onto a pair of silver wire hook recording electrodes and covered with liquid paraffin and then embedded in silicone sealant. 2.3. Experimental procedures In the first group of experiments, stimulation of the cervical vagus and bilateral cervical vagotomy were used separately to confirm (i) that the recording was from a vagal branch within the pancreas and (ii) the afferent nature of the discharge recorded from the isolated pancreatic nerve. Pancreatic vagal afferent discharge was recorded prior to and after systemic administration of cholecystokinin sulphated octapeptide (0.01–10 µg/kg, i.v.) and phenylbiguanide (0.01–10 µg/kg, i.v.) in the presence and absence of the cholecystokinin CCK1 receptor antagonists lorglumide (10 mg/kg, i.v.) or devazepide (0.5 mg/kg, i.v.) and the serotonin 5-HT3 receptor antagonists granisetron (1 mg/kg, i.v.) or MDL 72222 (0.1 mg/kg, i.v.). Cholecystokinin CCK1 and serotonin 5-HT3 receptor antagonists were administered 5–10 min prior to the intravenous injections of cholecystokinin or phenylbiguanide. Amplified and filtered (10–3000 Hz) pancreatic vagal afferent discharge was monitored using an oscilloscope (Tektronix Inc., Beaverton, Oregon, USA). All electrical signals were recorded using a computer-based data acquisition system (Power 1401, Cambridge Electronic Design Ltd, Cambridge, UK) running Spike 2 software (Cambridge Electronic Design Ltd, Cambridge, UK). The raw signals were rectified and normalized by subtracting the background noise determined at the end of the experiment from the raw signal and dividing it by the signal level measured at the beginning of the experiment and multiplying the result by 100. Pancreatic vagal afferent discharge is expressed in arbitrary units. The conduction velocity of the pancreatic nerve fibres was calculated by dividing the distance between the stimulation and recording sites (in meters) by the latency of the response (in seconds). 2.4. Statistical analysis All data are presented as mean ± S.E.M. The statistical significance of differences between the individual means was assessed and analysed using one way analysis of variance followed by Bonferroni
200
B.M. Mussa et al. / European Journal of Pharmacology 601 (2008) 198–206
Fig. 1. A representative example of pancreatic vagal afferent discharge responses to electrical stimulation of the cervical vagus nerve (200–800 µA, black arrows; n = 5). Stimuli (0.5 ms) were applied at 0.5 Hz and the evoked constant latency (84 ± 1.2 ms) nerve discharge responses ranged between 0.6 mV and 0.9 mV. Each trace represents the averaged response obtained after 100 applications of the cervical vagal stimulus. The mean conduction velocity was 0.83 ± 0.02 m/s (n = 5).
Multiple Comparisons Test. Probability values of P b 0.05 were considered statistically significant. 2.5. Materials Normal saline (0.9% w/v NaCl, Pharmacia & Upjohn Ltd., Perth, Australia) was used to dissolve or dilute all drugs used in this study.
Cholecystokinin sulphated octapeptide was obtained from American Peptide Co. (Sunnyvale, CA, USA). Phenylbiguanide was obtained from Aldrich Chemical Co. (St. Louis, MO, USA). Lorglumide sodium salt was obtained from Sigma-RBI (Sigma-Aldrich, St Louis, MO, U.S.A.). MDL 72222 ([(1S,5R)-8-methyl-8-azabicyclo[3.2.1]octan-3-yl] 3,5-dichlorobenzoate) was obtained from Tocris-Cookson (Bristol, UK) and granisetron was obtained from Beecham Pharmaceuticals (Cambridge, UK).
Fig. 2. Effects of cholecystokinin on pancreatic vagal afferent discharge prior to, and after bilateral cervical vagotomy. A, Effects of cholecystokinin (0.01–10 µg/kg, i.v.) and saline on mean arterial blood pressure, heart rate and pancreatic vagal afferent discharge prior to cervical vagotomy. The two top traces in each panel represent the normalized (in arbitrary units) and raw (in mV) discharge of the pancreatic nerve. B, Effects of cholecystokinin (0.01–10 µg/kg, i.v.) on mean arterial blood pressure, heart rate and pancreatic vagal afferent discharge after cervical vagotomy.
B.M. Mussa et al. / European Journal of Pharmacology 601 (2008) 198–206
201
Devazepide (Merck Research Laboratories, Rahway, NJ, U.S.A) was dissolved in dimethyl sulphoxide/polyethyleneglycol 400 (9:1) and then diluted with normal saline. 3. Results 3.1. Identification of the pancreatic vagus
Fig. 3. Group data for effects of cholecystokinin (0.01–10 µg/kg, i.v.) on pancreatic vagal afferent discharge prior to (open bars), and after (filled bars) bilateral cervical vagotomy. Cholecystokinin (0.1–10 µg/kg) before vagotomy produced significant increases in pancreatic vagal afferent discharge compared to saline, ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001. Cholecystokinin (0.1–10 µg/kg) after vagotomy produced significant increases in pancreatic vagal afferent discharge compared to saline, # P b 0.05, ## P b 0.01, ### P b 0.001. Mean responses to cholecystokinin before and after vagotomy are not significantly different (P N 0.05). All values are mean ± S.E.M. (n = 5).
The pancreas is innervated by sympathetic, parasympathetic and sensory nerves (Love et al., 2006). Therefore, it was essential to verify that the pancreatic nerve recording was vagal and afferent in nature. In the first group of experiments, electrical stimulation of the cervical vagus led to bradycardic responses confirming that the isolated nerve was a branch of the vagus. Intermittent electrical stimulation (0.5 Hz, 0.5 ms pulse width, 20–800 µA) of the cervical vagus nerve evoked constant latency bursts of activity in the pancreatic vagus nerve with a mean latency of 84 ± 1.2 ms, corresponding to a mean conduction velocity of 0.83 ± 0.02 m/s (n = 5; Fig. 1). Stimulation using currents ranging between 200–800 µA produced increases (0.6-0.9 mV, n = 5) in pancreatic vagal afferent discharge whereas stimulation with 20 µA did not evoke a pancreatic vagal afferent discharge response (Fig. 1).
Fig. 4. Effects of phenylbiguanide (0.01–10 µg/kg, i.v.) on pancreatic vagal afferent discharge prior to, and after bilateral cervical vagotomy. A, Effects of phenylbiguanide (0.01–10 µg/kg, i.v.) and saline on mean arterial blood pressure, heart rate and pancreatic vagal afferent discharge prior to cervical vagotomy. The two top traces represent the normalized (in arbitrary units) and raw (in mV) discharge of the pancreatic nerve. B, Effects of phenylbiguanide (0.01–10 µg/kg, i.v.) on mean arterial blood pressure, heart rate and pancreatic vagal afferent discharge after cervical vagotomy.
202
B.M. Mussa et al. / European Journal of Pharmacology 601 (2008) 198–206
The responses shown are averages obtained after 100 successive applications of the cervical vagal stimulus. 3.2. Effects of cervical vagotomy on pancreatic vagal afferent discharge responses to cholecystokinin and phenylbiguanide The effects of four doses of cholecystokinin (0.01–10 µg/kg) on pancreatic vagal afferent discharge were examined before and after cervical vagotomy. Cholecystokinin (0.1–10 µg/kg, i.v.) caused an immediate, dose-dependent and significant increase in pancreatic vagal afferent discharge (n = 5, P b 0.05). This increase lasted for ~20– 60 s after the administration of cholecystokinin. Pancreatic vagal afferent discharge was not changed after administration of the lowest dose of cholecystokinin (0.01 µg/kg) or normal saline, so this lowest dose was not further evaluated in this study (Figs. 2 and 3). Bilateral cervical vagotomy produced no change in the excitatory effects of cholecystokinin on pancreatic vagal afferent discharge suggesting that the discharge was largely recorded from vagal afferent fibres and not from efferent fibres (Figs. 2 and 3). The excitatory effects of cholecystokinin on pancreatic vagal afferent discharge were accompanied by depressor and bradycardic responses although these were not quantified in this study since the cardiovascular actions of cholecystokinin are unlikely to be related to the effects on pancreatic vagal afferent discharge. As shown in Figs. 4 and 5, the 1 and 10 µg/kg doses of phenylbiguanide produced a rapid, short-lasting (~ 5s) and significant increase in pancreatic vagal afferent discharge (n = 5, P b 0.05). In contrast, the 0.01 and 0.1 µg/kg doses of phenylbiguanide on pancreatic vagal afferent discharge produced no significant change in the pancreatic vagal afferent discharge. Therefore, only the effects of the 1 and 10 µg/kg doses of phenylbiguanide on pancreatic vagal afferent discharge were considered further in this study. Normal saline had no effect on pancreatic vagal afferent discharge. The responses of pancreatic vagal afferent discharge to phenylbiguanide (1 & 10 µg/kg) were not affected by cervical vagotomy indicating that the increased discharge was afferent in nature. Depressor and bradycardic responses to phenylbiguanide were observed along with its effects on pancreatic vagal afferent discharge but these responses were not quantified.
Fig. 6. Effects of the cholecystokinin CCK1 receptor antagonist lorglumide on the excitatory responses of pancreatic vagal afferent discharge to cholecystokinin and phenylbiguanide. A, Open and filled bars represent the responses to cholecystokinin (0.1–10 µg/kg, i.v.) prior to, and after lorglumide (10 mg/kg, i.v), respectively. B, Open and filled bars represent the responses to phenylbiguanide (1–10 µg/kg, i.v.) prior to, and after lorglumide (10 mg/kg, i.v.), respectively. All values are mean ± S.E.M. (n = 5). ⁎⁎P b 0.01 or ⁎⁎⁎P b 0.001 compared to pre-lorglumide responses.
3.3. Effects of cholecystokinin CCK1 receptor blockade on pancreatic vagal afferent discharge responses to cholecystokinin and phenylbiguanide Lorglumide (10 mg/kg, i.v.) significantly inhibited the excitatory action of cholecystokinin (0.1–10 µg/kg) on pancreatic vagal afferent
Fig. 5. Group data for effects of phenylbiguanide (0.01–10 µg/kg, i.v.) on pancreatic vagal afferent discharge prior to (open bars), and after (filled bars) bilateral cervical vagotomy. Phenylbiguanide (1–10 µg/kg) before vagotomy produced significant increases in pancreatic vagal afferent discharge compared to saline, ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001. Phenylbiguanide (1–10 µg/kg) after vagotomy produced significant increases in pancreatic vagal afferent discharge compared to saline, ## P b 0.01, ###P b 0.001. Mean responses to phenylbiguanide before and after vagotomy are not significantly different (P N 0.05). All values are mean ± S.E.M., n = 5.
Fig. 7. Effects of the cholecystokinin CCK1 receptor antagonist devazepide on the excitatory responses of pancreatic vagal afferent discharge to cholecystokinin and phenylbiguanide. A, Open and filled bars represent the responses to cholecystokinin (0.1–10 µg/kg, i.v.) prior to, and after devazepide (0.5 mg/kg, i.v.), respectively. B, Open and filled bars represent the responses to phenylbiguanide (1–10 µg/kg, i.v) prior to, and after devazepide (0.5 mg/kg, i.v.), respectively. All values are mean ± S.E.M. (n = 5). ⁎⁎P b 0.01 or ⁎⁎⁎P b 0.001 compared to pre-devazepide responses.
B.M. Mussa et al. / European Journal of Pharmacology 601 (2008) 198–206
203
discharge (n = 5, P b 0.05) (Fig. 6A). As shown in Fig. 6B, lorglumide (10 mg/kg, i.v.) produced a significant inhibition of the pancreatic vagal afferent discharge responses to the 1 µg/kg dose of phenylbiguanide (n = 5, P b 0.05) without affecting the response to the highest dose of phenylbiguanide (10 µg/kg). The excitatory effects of cholecystokinin (0.1–10 µg/kg) on pancreatic vagal afferent discharge were significantly inhibited after devazepide (0.5 mg/kg, i.v.) (n = 5, P b 0.05) (Fig. 7A). Devazepide (0.5 mg/kg, i.v.) treatment also produced a significant reduction in the phenylbiguanide (1 µg/kg)induced increases in pancreatic vagal afferent discharge (n = 5, P b 0.05) (Fig. 7B). 3.4. Effects of serotonin 5-HT3 receptor blockade on pancreatic vagal afferent discharge responses to phenylbiguanide and cholecystokinin Blockade of serotonin 5-HT 3 receptors with granisetron (10 mg/kg, i.v.) significantly attenuated the phenylbiguanide (10 µg/kg)-evoked excitation in pancreatic vagal afferent discharge (n = 5, P b 0.05) with no significant effect on the responses to the lower dose (1 µg/kg) (Fig. 8A). Granisetron (10 mg/kg, i.v.) did not alter the responses to cholecystokinin (0.1–10 µg/kg) (P N 0.05; Fig. 8B). The effects of the serotonin 5-HT3 receptor antagonist, MDL 72222, on phenylbiguanide- and cholecystokinin-induced responses in pancreatic vagal afferent discharge were also tested. MDL 72222 (0.1 mg/kg, i.v.) completely abolished the excitatory responses of pancreatic vagal afferent discharge to phenylbiguanide (1–10 µg/kg) (n = 5, P b 0.05) (Fig. 9A). Compared to granisetron, MDL 72222 was more effective in reducing the phenylbiguanide-induced excitatory effects on pancreatic vagal afferent discharge. However, it did not
Fig. 9. Effects of the serotonin 5-HT3 receptor antagonist MDL 72222 on the excitatory responses of pancreatic vagal afferent discharge to phenylbiguanide and cholecystokinin. A, Open and filled bars represent the responses to phenylbiguanide (1–10 µg/kg, i.v.) prior to, and after MDL 72222 (0.1 mg/kg, i.v.), respectively. B, Open and filled bars represent the responses to cholecystokinin sulphated octapeptide (0.1–10 µg/kg, i.v.) prior to, and after MDL 72222 (0.1 mg kg− 1, i.v.), respectively. All values are mean± S.E.M. (n = 5). ⁎⁎⁎P b 0.001 compared to pre-MDL 72222 responses.
affect the cholecystokinin-induced increase in pancreatic vagal afferent discharge (n = 5, P N 0.05) (Fig. 9B). In a separate group of experiments, the effects of pre-administration of cholecystokinin (10 µg/kg) on pancreatic vagal afferent discharge-responses to phenylbiguanide were tested. Phenylbiguanide (0.01 µg/kg) produced a significant increase in pancreatic vagal afferent discharge when administered ~ 5–8 min subsequent to cholecystokinin (10 µg/kg) administration. In contrast, the same dose of phenylbiguanide produced no increase in pancreatic vagal afferent discharge following saline administration (Figs. 10 and 11). As shown in Fig. 12, these excitatory effects of phenylbiguanide (0.01 µg/ kg) on pancreatic vagal afferent discharge were significantly reduced after administration of cholecystokinin CCK1 receptor antagonists (lorglumide and devazepide).
Fig. 8. Effects of the serotonin 5-HT3 receptor antagonist granisetron on the excitatory responses of pancreatic vagal afferent discharge to phenylbiguanide and cholecystokinin. A, Open and filled bars represent the responses to phenylbiguanide (1–10 µg/kg, i.v.) prior to, and after granisetron (1 mg /kg, i.v.), respectively. B, Open and filled bars represent the responses to cholecystokinin (0.1–10 µg/kg, i.v.) prior to, and after granisetron (1 mg/kg, i.v.), respectively. All values are mean ± S.E.M (n = 5). ⁎⁎⁎P b 0.001 compared to pregranisetron responses.
Fig. 10. Effects of pre-administration of cholecystokinin on pancreatic responses to phenylbiguanide. Open and filled bars represent the responses to phenylbiguanide (0.01 µg/kg, i.v.) prior to, and after administration of cholecystokinin (10 µg/kg, i.v.), respectively. All values are mean ± S.E.M. (n = 5). ⁎⁎P b 0.01.
204
B.M. Mussa et al. / European Journal of Pharmacology 601 (2008) 198–206
Fig. 11. Effects of cholecystokinin on pancreatic vagal afferent discharge-responses to phenylbiguanide. A, Effects of phenylbiguanide (0.01 µg/kg, i.v.) on mean arterial blood pressure, heart rate and pancreatic vagal afferent discharge after administration of cholecystokinin (10 µg/kg, i.v.). The two top traces in each panel represent the normalized (in arbitrary units) and raw (in mV) discharge of the pancreatic nerve. B, Effects of phenylbiguanide (0.1 µg/kg, i.v.) on mean arterial blood pressure, heart rate and pancreatic vagal afferent discharge after administration of saline.
4. Discussion
Fig. 12. Effects of the cholecystokinin CCK1 receptor antagonists lorglumide and devazepide on the excitatory responses of pancreatic vagal afferent discharge to phenylbiguanide after administration of cholecystokinin. Open and filled bars represent effects of phenylbiguanide (0.01 µg/kg, i.v.) on pancreatic vagal afferent discharge prior to and after cholecystokinin CCK1 receptor antagonists (devazepide, 0.5 mg/kg, i.v. and lorglumide, 10 mg/kg, i.v). All values are mean ± S.E.M. (n = 5). ⁎⁎⁎P b 0.001.
Peripheral vagal afferents sense a broad range of chemical, physical and hormonal signals (Davison and Clarke, 1988; Marik and Code, 1975; Schwartz and Moran, 1998; Schwartz et al., 1995). Cholecystokinin is released from the enteroendocrine cells of the duodenum and mediates various digestive functions via activation of its receptors on peripheral vagal afferents (Adler et al., 1991; Blackshaw and Grundy, 1990; Li and Owyang, 1994; Simasko and Ritter, 2003; Viard et al., 2007). In agreement with a previous report, the results of the present study have demonstrated that pancreatic vagal afferents are highly sensitive to cholecystokinin (Niijima, 1981). Compared to Niijima's study, cholecystokinin produced more rapid and larger increases in pancreatic vagal afferent discharge and this could be due to several differences between the two studies including: (i) cholecystokinin dose range, (ii) route of cholecystokinin administration and (iii) the branch of the pancreatic vagus nerve that was used to record the afferent discharge. In the present study, intravenous administration of
B.M. Mussa et al. / European Journal of Pharmacology 601 (2008) 198–206
cholecystokinin doses in random order increased pancreatic vagal afferent discharge significantly in a dose-related manner. The responses to cholecystokinin did not change after cervical vagotomy indicating that the pancreatic vagal nerve discharge was afferent in nature. It has generally been assumed that cholecystokinin influences pancreatic secretory function via a vago-vagal reflex (Li and Owyang, 1993, 1994). Since the site of action of cholecystokinin on medullary presympathetic vasomotor neurons is on sub-diaphragmatic vagal afferents, it is conceivable that pancreatic vagal afferents are also involved in this response (Verberne and Sartor, 2004). Similarly, the depressor response to cholecystokinin is believed to be due to an inhibitory action of cholecystokinin on sympathetic vasomotor outflow (Sartor and Verberne, 2003, 2002). An important finding of this study is that the cholecystokinin CCK1 receptor mediates cholecystokinin-induced activation of pancreatic vagal afferents. This was demonstrated by the reduction in the excitatory effects of cholecystokinin on pancreatic vagal afferent discharge by pre-treatment with lorglumide or devazepide. On the other hand, blockade of serotonin 5-HT3 receptors produced no change in the cholecystokinin-evoked excitatory effects on pancreatic vagal afferent discharge suggesting that the effects of cholecystokinin are not dependent on serotonin 5-HT3 receptors. 5-HT is an important modulator of pancreatic secretion that exerts its effects via activation of duodenal mucosal vagal afferents (Li et al., 2000). The results of the present study suggest that pancreatic vagal afferents are another peripheral site at which serotonin 5-HT3 receptor agonists may act. The effects of four doses (0.01–10 µg/kg) of phenylbiguanide on pancreatic vagal afferent discharge were examined but only two doses (1 & 10 µg/kg) have produced noticeable effects. This study provides direct evidence for the involvement of serotonin 5-HT3 receptors in the effects of 5-HT on pancreatic vagal afferent discharge. Blockade of serotonin 5-HT3 receptors with two different antagonists, granisetron and MDL 72222, significantly reduced the pancreatic vagal afferent discharge response to the highest dose of phenylbiguanide (10 µg/kg). In contrast, the 1 µg/kg dose of phenylbiguanide was slightly reduced after granisetron but significantly attenuated after MDL72222. The latter seems to be more effective in inhibiting serotonin 5-HT3 receptor agonist effects on pancreatic vagal afferent discharge. Interestingly, cholecystokinin CCK1 receptor blockade with either lorglumide or devazepide significantly attenuated the responses to the low (1 µg/kg) but not the high (10 µg/kg) dose of phenylbiguanide. This novel finding suggests that phenylbiguanide acts, in part, via an interaction with cholecystokinin CCK1 receptors. Additional experiments have validated this hypothesis by showing that cholecystokinin CCK1 receptors antagonists were able to block the excitatory effects of phenylbiguanide (0.01 µg/kg) on pancreatic vagal afferent discharge which were probably due to an interaction between cholecystokinin CCK1 and serotonin 5-HT3 receptors. A similar interaction was observed in a study which demonstrated that blockade of cholecystokinin CCK1 receptors reduced the 5-HT-induced contractile responses of the guinea-pig ileum (Fortuño et al., 1999). In contrast, previous reports have shown that serotonin 5-HT3 receptors contribute to some of the effects of cholecystokinin including satiety, excitatory effects on vagal afferent neurons recorded from the nodose ganglion and the inhibitory actions on presympathetic vasomotor neurons of the rostral ventrolateral medulla (Aja, 2006; Hayes et al., 2004; Li et al., 2004; Saita and Verberne, 2003). Given the results of the present study, it is possible that some of the actions of serotonin 5-HT3 receptor antagonists are centrally-mediated. In conclusion, the present findings demonstrate that cholecystokinin and phenylbiguanide are both potent activators of pancreatic vagal afferents. Additionally, we have confirmed that cholecystokinin CCK1 and serotonin 5-HT3 receptors are mediators of the excitatory effects of cholecystokinin and 5-HT, respectively, on pancreatic vagal
205
afferent discharge. These receptors interact in a fashion that may influence pancreatic sensory function. The presence of cholecystokinin CCK1 and serotonin 5-HT3 receptors in this reflex pathway may be important for control of pancreatic exocrine and endocrine secretion. Conflict of Interest The authors state no conflict of interest. Acknowledgement This work was supported by the Austin Hospital Medical Research Foundation. References Adler, G., Beglinger, C., Braun, U., Reinshagen, M., Koop, I., Schafmayer, A., Rovati, L., Arnold, R., 1991. Interaction of the cholinergic system and cholecystokinin in the regulation of endogenous and exogenous stimulation of pancreatic secretion in humans. Gastroenterology 100, 537–543. Aja, S., 2006. Serotonin-3 receptors in gastric mechanisms of cholecystokinin-induced satiety. Am. J. Physiol. 291, R112–R114. Blackshaw, L.A., Grundy, D., 1990. Effects of cholecystokinin (cholecystokinin sulphated octapeptide-8) on two classes of gastroduodenal vagal afferent fibre. J. Auton. Nerv. Syst. 31, 191–201. Chang, T.M., Chey, W.Y., 2001. Neurohormonal control of exocrine pancreas. Curr. Opin. Gastroenterol. 17, 416–425. Chey, W.Y., Chang, T., 2001. Neural hormonal regulation of exocrine pancreatic secretion. Pancreatology 1, 320–335. Davison, J.S., Clarke, G.D., 1988. Mechanical properties and sensitivity to cholecystokinin sulphated octapeptide of vagal gastric slowly adapting mechanoreceptors. Am. J. Physiol. 255, G55–61. Fortuño, A., Ballaz, S., Del Río, J., Barber, A., 1999. Cholecystokinin sulphated octapeptide-mediated response in the activation of 5-HT receptor types in the guinea-pig ileum. J. Physiol. Biochem. 55, 85–92. Hayes, M.R., Moore, R.L., Shah, S.M., Covasa, M., 2004. 5-HT3 receptors participate in cholecystokinin sulphated octapeptide-induced suppression of food intake by delaying gastric emptying. Am. J. Physiol. 287, R817–R823. Konturek, S.J., Tasler, J., Obtulowicz, W., 1979. Effect of atropine on pancreatic responses endogenous and exogenous cholecystokinin. Am. J. Dig. Dis. 17, 911–917. Li, Y., Owyang, C., 1993. Vagal afferent pathway mediates physiological action of cholecystokinin on pancreatic enzyme secretion. J. Clin. Invest. 92, 418–424. Li, Y., Owyang, C., 1994. Endogenous cholecystokinin stimulates pancreatic enzyme secretion via vagal afferent pathway in rats. Gastroenterology 107, 525–531. Li, Y., Owyang, C., 1996. Pancreatic secretion evoked by cholecystokinin and noncholecystokinin-dependent duodenal stimuli via vagal afferent fibres in the rat. J. Physiol. (London) 494, 773–782. Li, Y., Hao, Y., Owyang, C., 1997. High-affinity cholecystokinin sulphated octapeptide-A receptors on the vagus nerve mediate cholecystokinin sulphated octapeptidestimulated pancreatic secretion in rats. Am. J. Physiol. 273, G679–G685. Li, P., Chang, T.M., Chey, W.Y., 1998. Secretin inhibits gastric acid secretion via a vagal afferent pathway in rats. Am. J. Physiol. 275, G22–G28. Li, Y., Zhu, J., Owyang, C.,1999. Electrical physiological evidence for high and low-affinity vagal cholecystokinin sulphated octapeptide-A receptors. Am. J. Physiol. 277, R469–R477. Li, Y., Hao, Y., Zhu, J., Owyang, C., 2000. Serotonin released from intestinal enterochromaffin cells mediates luminal non-cholecystokinin-stimulated pancreatic secretion in rats. Gastroenterology 118, 1197–1207. Li, J.P., Chang, T.M., Chey, W.Y., 2001a. Roles of 5-HT receptors in the release and action of secretin on pancreatic secretion in rats. Am. J. Physiol. 280, G523–G538. Li, Y., Wu, X.Y., Zhu, J.X., Owyang, C., 2001b. Intestinal serotonin acts as paracrine substance to mediate pancreatic secretion stimulated by luminal factors. Am. J. Physiol. 281, G916–G923. Li, Y., Wu, X.Y., Owyang, C., 2004. Serotonin and cholecystokinin synergistically stimulate rat vagal primary afferent neurones. J. Physiol. 55, 651–662. Li, Y., Wu, X., Yao, H., Owyang, C., 2005. Secretin activates vagal primary afferent neurons in the rat: evidence from electrophysiological and immunohistochemical studies. Am. J. Physiol. 289, G745–G752. Liao, Z., Li, Z.S., Lu, Y., Wang, W.Z., 2005. Glutamate receptors within the nucleus of solitary tract contribute to pancreatic secretion stimulated by intraduodenal hypertonic saline. Auton. Neurosci. 120, 62–67. Love, J.A., Yi, E., Smith, T.G., 2006. Autonomic pathways regulating pancreatic exocrine secretion. Auton. Neurosci. 16, 1–16. Marik, F., Code, C.F., 1975. Control of the interdigestive myoelectric activity in dogs by the vagus nerves and pentagastrin. Gastroenterology 69, 387–395. Moran, T.H., Smith, G.P., Hostetler, A.M., McHugh, P.R., 1987. Transport of cholecystokinin (cholecystokinin sulphated octapeptide) binding sites in subdiaphragmatic vagal branches. Brain Res. 415, 149–152. Moriarty, P., Dimaline, R., Thompson, D.G., Dockray, G.J., 1997. Characterization of cholecystokininA and cholecystokininB receptors expressed by vagal afferent neurons. Neuroscience 79, 905–913. Mussa, B., Verberne, A.J., 2008. Activation of the dorsal vagal nucleus increases pancreatic exocrine secretion in the rat. Neurosci. Lett. 433, 71–76.
206
B.M. Mussa et al. / European Journal of Pharmacology 601 (2008) 198–206
Niebergall-Roth, E., Singer, M.V., 2001. Central and peripheral neural control of pancreatic exocrine secretion. J. Physiol. Pharmacol. 52, 523–538. Niijima, A., 1981. Visceral afferents and metabolic function. Diabetologia 20, 325–330. Owyang, C., 1996. Physiological mechanisms of cholecystokinin action on pancreatic secretion. Am. J. Pysiol. 34, G1–G7. Owyang, C., Logsdon, C.D., 2004. New insights into neurohormonal regulation of pancreatic secretion. Gastroenterology 127, 957–969. Raybould, H.E., Glatzle, J., Robin, C., Meyer, J.H., Phan, T., Wong, H., Sternini, C., 2003. Expression of 5-HT3 receptors by extrinsic duodenal afferents contribute to intestinal inhibition of gastric emptying. Am. J. Physiol. 284, G367–G372. Saita, M., Verberne, A.J., 2003. Roles for cholecystokinin sulphated octapeptide1 and 5-HT3 receptors in the effects of cholecystokinin sulphated octapeptide on presympathetic vasomotor neuronal discharge in the rat. Br. J. Pharmacol. 139, 415–423. Sartor, D.M., Verberne, J.M.J., 2002. Cholecystokinin selectively affects presympathetic vasomotor neurons and sympathetic vasomotor outflow. Am. J. Physiol. 280, R1174–R1184. Sartor, D.M., Verberne, A.J.M., 2003. Phenotypic identification of rat rostroventrolateral medullary presympathetic vasomotor neurons inhibited by exogenous cholecystokinin. J. Comp. Neurol. 456, 467–479. Schwartz, G.J., Moran, T.H., 1998. Duodenal nutrient exposure elicits nutrient-specific gut motility and vagal afferent signals in rat. Am. J. Physiol. 274, R1236–R1242. Schwartz, G.J., Tougas, G., Moran, T.H., 1995. Integration of vagal afferent responses to duodenal loads and exogenous cholecystokinin sulphated octapeptide in rats. Peptides 16, 707–711.
Simasko, M.S., Ritter, C.R., 2003. Cholecystokinin activates both A- and C-type vagal afferent neurons. Am. J. Physiol. 285, G1204–G1213. Soudah, H.C., Lu, Y., Hasler, W.L., Owyang, C., 1992. Cholecystokinin at physiological levels evokes pancreatic enzyme secretion via a cholinergic pathway. Am. J. Physiol. 263, G102–G107. Travagli, R.A., Gillis, R.A., Rossiter, C.D., Vicini, S., 1991. Glutamate and GABA-mediated synaptic currents in neurons of the rat dorsal motor nucleus of the vagus. Am. J. Physiol.: Gastrointest. Liver Physiol. 260. Travagli, R.A., Hermann, G.E., Browning, K.N., Rogers, R.C., 2006. Brainstem circuits regulating gastric function. Annu. Rev. Physiol. 68, 279–305. Verberne, A.J.M., Sartor, D.M., 2004. Cholecystokinin sulphated octapeptide-induced inhibition of presympathetic vasomotor neurons: dependence on subdiaphragmatic vagal afferents and central NMDA receptors in the rat. Am. J. Physiol., Regul. Integr. Comp. Physiol. 287, R809–R816. Viard, E., Zheng, Z., Wan, S., Travagli, R.A., 2007. Vagally mediated, nonparacrine effects of cholecystokinin-8 s on rat pancreatic exocrine secretion. Am. J. Physiol. 293, G493–G500. Zarbin, M.A., Wamsley, J.K., Innis, R.B., Kuhar, M.J., 1981. Cholecystokinin receptors: presence and axonal flow in the rat vagus nerve. Life. Sci. 29, 697–705.
Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / e j p h a r
Endocrine Pharmacology
Activation of cholecystokinin (CCK1) and serotonin (5-HT3) receptors increases the discharge of pancreatic vagal afferents Bashair M. Mussa, Daniela M. Sartor, Anthony J.M. Verberne ⁎ University of Melbourne, Department of Medicine, Clinical Pharmacology and Therapeutics Unit, Austin Health, Heidelberg 3084, Victoria, Australia
a r t i c l e
i n f o
Article history: Received 24 October 2008 Accepted 3 November 2008 Available online 9 November 2008 Keywords: Pancreatic vagal afferent Cholecystokinin CCK1 receptor Serotonin 5-HT3 receptor
a b s t r a c t Cholecystokinin and serotonin are released from the gastrointestinal tract in response to the products of digestion and play critical roles in mediating pancreatic secretion via vago-vagal reflex pathways. This study was designed to investigate the effects of activation of cholecystokinin CCK1 and serotonin (5-hydroxytryptamine, 5-HT) 5-HT3 receptors on pancreatic vagal afferent discharge and to determine whether there is an interaction between these receptors. Male Sprague Dawley rats anaesthetised with isoflurane (1.5%/100% O2) were used in all experiments. The effects of systemic administration of cholecystokinin and the serotonin 5-HT3 receptor agonist phenylbiguanide on pancreatic vagal afferent discharge were recorded before and after administration of cholecystokinin CCK1 and serotonin 5-HT3 receptor antagonists. Cholecystokinin (0.1–10 µg/kg, i.v.) and phenylbiguanide (1 and 10 µg/kg, i.v.) increased pancreatic vagal afferent discharge dose-dependently. Cholecystokinin CCK1 receptor antagonists, lorglumide (10 mg/kg, i.v.) and devazepide (0.5 mg/kg, i.v.), reduced cholecystokinin- and phenylbiguanide-induced increases in pancreatic vagal afferent discharge significantly (n = 5, P b 0.05). On the other hand, serotonin 5-HT3 receptor blockade with granisetron (1 mg/kg, i.v.) or MDL72222 ([(1S,5R)-8-methyl-8-azabicyclo[3.2.1]octan-3-yl] 3,5-dichlorobenzoate; 0.1 mg/kg, i.v.) inhibited the pancreatic vagal afferent discharge responses to phenylbiguanide but not those to cholecystokinin. This study has confirmed that cholecystokinin and phenylbiguanide activate pancreatic vagal afferent discharge via activation of cholecystokinin CCK1 and serotonin 5-HT3 receptors, respectively. In addition, it has demonstrated that (i) the serotonin 5-HT3 agonist phenylbiguanide acts partly via an interaction with cholecystokinin CCK1 receptors, and (ii) the actions of cholecystokinin are not dependent on serotonin 5-HT3 receptor activation. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Classically, the control of pancreatic secretion has been viewed as being dependent on two separate mechanisms: (i) the release of gastrointestinal hormones into the circulation and (ii) a vago-vagal reflex pathway. However, a recent notion has suggested that these two mechanisms act synergistically and proposes that activation of a vagovagal reflex by gastrointestinal hormones such as cholecystokinin and serotonin (5-hydroxytryptamine, 5-HT) mediates a considerable portion of pancreatic secretion (Li et al., 2000; Li and Owyang, 1994, 1996; Li et al., 2001b). The vago-vagal reflex pathway that regulates pancreatic secretion consists of three major components: (i) duodenal and pancreatic vagal afferents which originate from pseudo-bipolar neurons in the nodose ganglion and project in a bidirectional fashion to the nucleus of the solitary tract in the dorsal medulla and peripherally to the duodenum and pancreas, (ii) visceroceptive neurons in the nucleus of solitary tract that integrate the vagal afferent input, and (iii) pancreatic preganglionic
⁎ Corresponding author. Tel.: +61 3 94965978; fax: +61 3 94593510. E-mail address: [email protected] (A.J.M. Verberne). 0014-2999/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2008.11.007
vagal neurons which project mainly from the dorsal vagal nucleus. More detail regarding the role of these pathways in control of pancreatic secretion can be found in recent reviews (Love et al., 2006; NiebergallRoth and Singer, 2001). The vago-vagal reflex model suggests that gastrointestinal hormones such as cholecystokinin and 5-HT activate receptors on vagal afferents which terminate onto and activate neurons within the nucleus of the solitary tact via glutamate receptors (Liao et al., 2005). These neurons, in turn, influence pancreatic preganglionic neurons projecting from the dorsal vagal nucleus and modulate the vagal output to the pancreas via glutamatergic and GABAergic inputs (Love et al., 2006; Mussa and Verberne, 2008; Travagli et al., 1991, 2006). It is of interest to further explore the actions of cholecystokinin and 5-HT on pancreatic vagal afferents since this represents an integral part of the vago-vagal reflex that contributes to control of pancreatic secretion. While several gastrointestinal hormones are known to contribute to regulation of pancreatic secretion (Chang and Chey, 2001; Chey and Chang, 2001), cholecystokinin and 5-HT are believed to play major roles (Li et al., 2001a, 2000, 2001b; Owyang, 1996; Owyang and Logsdon, 2004). Several lines of evidence support the view that cholecystokinin and 5-HT act in a paracrine fashion on duodenal vagal
B.M. Mussa et al. / European Journal of Pharmacology 601 (2008) 198–206
afferents to influence pancreatic secretion in humans, rats and dogs (Konturek et al., 1979; Li and Owyang, 1993, 1996; Li et al., 2001b; Soudah et al., 1992). Additionally, secretin is one of the principal hormones that regulates secretion of bicarbonate through a direct action on pancreatic acini and perhaps also via activation of vagal afferent pathways (Li et al., 1998, 2005). In vitro and in vivo studies have confirmed that cholecystokinin receptors present on duodenal vagal afferents are predominantly the cholecystokinin CCK1 receptor subtype (Moran et al., 1987; Moriarty et al.,1997; Zarbin et al., 1981). There is electrophysiological evidence for high and low affinity states of the cholecystokinin CCK1 receptor, although the high-affinity receptor appears to be more important for pancreatic secretion (Li et al., 1997, 1999). Duodenal vagal afferents express a high density of serotonin 5-HT3 receptors and these are distributed widely throughout the gastrointestinal tract (Li et al., 2001a; Raybould et al., 2003). Cholecystokinin and 5-HT are potent stimulators of cholecystokinin CCK1 and serotonin 5-HT3 receptors on vagal afferents, respectively, and activate vago-vagal reflexes that are involved in regulation of several digestive processes including pancreatic secretion (Li et al., 2001a, 1997, 2000). Vagal afferents convey sensory information from various visceral organs including the duodenum and the pancreas. Thus, it is likely that vagal afferents arising from the latter may also be involved in regulation of pancreatic secretion. We hypothesised that cholecystokinin and 5-HT may activate the pancreatic vagal afferents by acting on cholecystokinin CCK1 and serotonin 5-HT3 receptors on these afferents, respectively. Although an earlier study by Niijma had shown that intracarotid injection of cholecystokinin and 5-HT produced an increase in pancreatic vagal afferent discharge, the receptors involved were not identified (Niijima, 1981). In addition, several studies have identified an interaction between cholecystokinin CCK1 and serotonin 5-HT3 receptors (Aja, 2006; Hayes et al., 2004; Li et al., 2004; Saita and Verberne, 2003). In these studies, serotonin 5-HT3 receptors were shown to mediate part of the inhibitory actions of cholecystokinin on food intake or on the discharge of medullary presympathetic vasomotor neurons. In view of these findings, the current study was designed to (i) investigate the effects of systemic administration of cholecystokinin and the serotonin 5-HT3 receptor agonist phenylbiguanide on pancreatic vagal afferent discharge specifically, (ii) to confirm that these effects are mediated by cholecystokinin CCK1 and serotonin 5HT3 receptors, respectively, and (iii) to determine whether there is an interaction between these receptors. We demonstrated that cholecystokinin and phenylbiguanide activate pancreatic vagal afferent discharge via cholecystokinin CCK1 and serotonin 5-HT3 receptors, respectively, and that there is an interaction between these receptors at the level of the pancreatic vagal afferents. 2. Methods and materials 2.1. Animal preparations All animal experimental protocols were conducted according to the guidelines of the Ethical Review Committee of the Austin and Repatriation Medical Centre (Heidelberg, Victoria, Australia) and the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Experiments were performed on 40 male Sprague Dawley rats weighing between 250–350 g obtained from the Animal Resource Centre (Perth, Western Australia). Rats were housed in a temperaturecontrolled environment with a 12 hour light-dark cycle and given free access to water and chow. They were initially anaesthetised using isoflurane vapour and were then tracheostomized to allow artificial ventilation with 100% O2 (1 ml/100 g body weight, 50–60 breaths/ min) containing 1.5–1.7% isoflurane. The body temperature was maintained at 36–38 °C throughout the experiment using a servocontrolled heat pad and the adequacy of anaesthesia was assessed by
199
absence of a withdrawal reflex to paw pinch as well as the absence of an eye blink response to gentle probing of the cornea. The adequacy of anaesthesia was re-assessed every 15 min and the anaesthetic vapour concentration was increased when necessary. The left carotid artery and left jugular vein were cannulated to monitor the arterial blood pressure and heart rate and to allow intravenous administration of drugs, respectively. 2.2. Isolation of the cervical and pancreatic vagus A small incision was made along the right side of the neck to expose the right cervical vagus nerve which was dissected free from the sheath and connective tissues. The intact nerve was placed onto a pair of silver wire hook electrodes and covered with liquid paraffin and then embedded in silicone sealant (Kwik-Cast, World Precision Instruments, Sarasota, FL, USA). Effectiveness of the cervical vagal stimulation was confirmed by observation of an immediate bradycardia and depressor response following application of electrical stimulation (5 Hz, 0.5 ms pulse width, 500 µA, 20s) to the vagus. A midline incision was made through the abdomen to gain access to the pancreas. The spleen was retracted caudally and laterally and the stomach was retracted contralaterally to expose the splenic segment of the pancreas. The pancreatic vagus nerve was traced along the coeliac branch of the posterior subdiaphragmatic vagal trunk which enters the pancreas tissue via the splenic artery. It was dissected carefully at about 0.5 cm from the point at which the splenic artery itself branches off from the coeliac artery. The pancreatic vagus nerve was isolated from the surrounding connective tissue and placed onto a pair of silver wire hook recording electrodes and covered with liquid paraffin and then embedded in silicone sealant. 2.3. Experimental procedures In the first group of experiments, stimulation of the cervical vagus and bilateral cervical vagotomy were used separately to confirm (i) that the recording was from a vagal branch within the pancreas and (ii) the afferent nature of the discharge recorded from the isolated pancreatic nerve. Pancreatic vagal afferent discharge was recorded prior to and after systemic administration of cholecystokinin sulphated octapeptide (0.01–10 µg/kg, i.v.) and phenylbiguanide (0.01–10 µg/kg, i.v.) in the presence and absence of the cholecystokinin CCK1 receptor antagonists lorglumide (10 mg/kg, i.v.) or devazepide (0.5 mg/kg, i.v.) and the serotonin 5-HT3 receptor antagonists granisetron (1 mg/kg, i.v.) or MDL 72222 (0.1 mg/kg, i.v.). Cholecystokinin CCK1 and serotonin 5-HT3 receptor antagonists were administered 5–10 min prior to the intravenous injections of cholecystokinin or phenylbiguanide. Amplified and filtered (10–3000 Hz) pancreatic vagal afferent discharge was monitored using an oscilloscope (Tektronix Inc., Beaverton, Oregon, USA). All electrical signals were recorded using a computer-based data acquisition system (Power 1401, Cambridge Electronic Design Ltd, Cambridge, UK) running Spike 2 software (Cambridge Electronic Design Ltd, Cambridge, UK). The raw signals were rectified and normalized by subtracting the background noise determined at the end of the experiment from the raw signal and dividing it by the signal level measured at the beginning of the experiment and multiplying the result by 100. Pancreatic vagal afferent discharge is expressed in arbitrary units. The conduction velocity of the pancreatic nerve fibres was calculated by dividing the distance between the stimulation and recording sites (in meters) by the latency of the response (in seconds). 2.4. Statistical analysis All data are presented as mean ± S.E.M. The statistical significance of differences between the individual means was assessed and analysed using one way analysis of variance followed by Bonferroni
200
B.M. Mussa et al. / European Journal of Pharmacology 601 (2008) 198–206
Fig. 1. A representative example of pancreatic vagal afferent discharge responses to electrical stimulation of the cervical vagus nerve (200–800 µA, black arrows; n = 5). Stimuli (0.5 ms) were applied at 0.5 Hz and the evoked constant latency (84 ± 1.2 ms) nerve discharge responses ranged between 0.6 mV and 0.9 mV. Each trace represents the averaged response obtained after 100 applications of the cervical vagal stimulus. The mean conduction velocity was 0.83 ± 0.02 m/s (n = 5).
Multiple Comparisons Test. Probability values of P b 0.05 were considered statistically significant. 2.5. Materials Normal saline (0.9% w/v NaCl, Pharmacia & Upjohn Ltd., Perth, Australia) was used to dissolve or dilute all drugs used in this study.
Cholecystokinin sulphated octapeptide was obtained from American Peptide Co. (Sunnyvale, CA, USA). Phenylbiguanide was obtained from Aldrich Chemical Co. (St. Louis, MO, USA). Lorglumide sodium salt was obtained from Sigma-RBI (Sigma-Aldrich, St Louis, MO, U.S.A.). MDL 72222 ([(1S,5R)-8-methyl-8-azabicyclo[3.2.1]octan-3-yl] 3,5-dichlorobenzoate) was obtained from Tocris-Cookson (Bristol, UK) and granisetron was obtained from Beecham Pharmaceuticals (Cambridge, UK).
Fig. 2. Effects of cholecystokinin on pancreatic vagal afferent discharge prior to, and after bilateral cervical vagotomy. A, Effects of cholecystokinin (0.01–10 µg/kg, i.v.) and saline on mean arterial blood pressure, heart rate and pancreatic vagal afferent discharge prior to cervical vagotomy. The two top traces in each panel represent the normalized (in arbitrary units) and raw (in mV) discharge of the pancreatic nerve. B, Effects of cholecystokinin (0.01–10 µg/kg, i.v.) on mean arterial blood pressure, heart rate and pancreatic vagal afferent discharge after cervical vagotomy.
B.M. Mussa et al. / European Journal of Pharmacology 601 (2008) 198–206
201
Devazepide (Merck Research Laboratories, Rahway, NJ, U.S.A) was dissolved in dimethyl sulphoxide/polyethyleneglycol 400 (9:1) and then diluted with normal saline. 3. Results 3.1. Identification of the pancreatic vagus
Fig. 3. Group data for effects of cholecystokinin (0.01–10 µg/kg, i.v.) on pancreatic vagal afferent discharge prior to (open bars), and after (filled bars) bilateral cervical vagotomy. Cholecystokinin (0.1–10 µg/kg) before vagotomy produced significant increases in pancreatic vagal afferent discharge compared to saline, ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001. Cholecystokinin (0.1–10 µg/kg) after vagotomy produced significant increases in pancreatic vagal afferent discharge compared to saline, # P b 0.05, ## P b 0.01, ### P b 0.001. Mean responses to cholecystokinin before and after vagotomy are not significantly different (P N 0.05). All values are mean ± S.E.M. (n = 5).
The pancreas is innervated by sympathetic, parasympathetic and sensory nerves (Love et al., 2006). Therefore, it was essential to verify that the pancreatic nerve recording was vagal and afferent in nature. In the first group of experiments, electrical stimulation of the cervical vagus led to bradycardic responses confirming that the isolated nerve was a branch of the vagus. Intermittent electrical stimulation (0.5 Hz, 0.5 ms pulse width, 20–800 µA) of the cervical vagus nerve evoked constant latency bursts of activity in the pancreatic vagus nerve with a mean latency of 84 ± 1.2 ms, corresponding to a mean conduction velocity of 0.83 ± 0.02 m/s (n = 5; Fig. 1). Stimulation using currents ranging between 200–800 µA produced increases (0.6-0.9 mV, n = 5) in pancreatic vagal afferent discharge whereas stimulation with 20 µA did not evoke a pancreatic vagal afferent discharge response (Fig. 1).
Fig. 4. Effects of phenylbiguanide (0.01–10 µg/kg, i.v.) on pancreatic vagal afferent discharge prior to, and after bilateral cervical vagotomy. A, Effects of phenylbiguanide (0.01–10 µg/kg, i.v.) and saline on mean arterial blood pressure, heart rate and pancreatic vagal afferent discharge prior to cervical vagotomy. The two top traces represent the normalized (in arbitrary units) and raw (in mV) discharge of the pancreatic nerve. B, Effects of phenylbiguanide (0.01–10 µg/kg, i.v.) on mean arterial blood pressure, heart rate and pancreatic vagal afferent discharge after cervical vagotomy.
202
B.M. Mussa et al. / European Journal of Pharmacology 601 (2008) 198–206
The responses shown are averages obtained after 100 successive applications of the cervical vagal stimulus. 3.2. Effects of cervical vagotomy on pancreatic vagal afferent discharge responses to cholecystokinin and phenylbiguanide The effects of four doses of cholecystokinin (0.01–10 µg/kg) on pancreatic vagal afferent discharge were examined before and after cervical vagotomy. Cholecystokinin (0.1–10 µg/kg, i.v.) caused an immediate, dose-dependent and significant increase in pancreatic vagal afferent discharge (n = 5, P b 0.05). This increase lasted for ~20– 60 s after the administration of cholecystokinin. Pancreatic vagal afferent discharge was not changed after administration of the lowest dose of cholecystokinin (0.01 µg/kg) or normal saline, so this lowest dose was not further evaluated in this study (Figs. 2 and 3). Bilateral cervical vagotomy produced no change in the excitatory effects of cholecystokinin on pancreatic vagal afferent discharge suggesting that the discharge was largely recorded from vagal afferent fibres and not from efferent fibres (Figs. 2 and 3). The excitatory effects of cholecystokinin on pancreatic vagal afferent discharge were accompanied by depressor and bradycardic responses although these were not quantified in this study since the cardiovascular actions of cholecystokinin are unlikely to be related to the effects on pancreatic vagal afferent discharge. As shown in Figs. 4 and 5, the 1 and 10 µg/kg doses of phenylbiguanide produced a rapid, short-lasting (~ 5s) and significant increase in pancreatic vagal afferent discharge (n = 5, P b 0.05). In contrast, the 0.01 and 0.1 µg/kg doses of phenylbiguanide on pancreatic vagal afferent discharge produced no significant change in the pancreatic vagal afferent discharge. Therefore, only the effects of the 1 and 10 µg/kg doses of phenylbiguanide on pancreatic vagal afferent discharge were considered further in this study. Normal saline had no effect on pancreatic vagal afferent discharge. The responses of pancreatic vagal afferent discharge to phenylbiguanide (1 & 10 µg/kg) were not affected by cervical vagotomy indicating that the increased discharge was afferent in nature. Depressor and bradycardic responses to phenylbiguanide were observed along with its effects on pancreatic vagal afferent discharge but these responses were not quantified.
Fig. 6. Effects of the cholecystokinin CCK1 receptor antagonist lorglumide on the excitatory responses of pancreatic vagal afferent discharge to cholecystokinin and phenylbiguanide. A, Open and filled bars represent the responses to cholecystokinin (0.1–10 µg/kg, i.v.) prior to, and after lorglumide (10 mg/kg, i.v), respectively. B, Open and filled bars represent the responses to phenylbiguanide (1–10 µg/kg, i.v.) prior to, and after lorglumide (10 mg/kg, i.v.), respectively. All values are mean ± S.E.M. (n = 5). ⁎⁎P b 0.01 or ⁎⁎⁎P b 0.001 compared to pre-lorglumide responses.
3.3. Effects of cholecystokinin CCK1 receptor blockade on pancreatic vagal afferent discharge responses to cholecystokinin and phenylbiguanide Lorglumide (10 mg/kg, i.v.) significantly inhibited the excitatory action of cholecystokinin (0.1–10 µg/kg) on pancreatic vagal afferent
Fig. 5. Group data for effects of phenylbiguanide (0.01–10 µg/kg, i.v.) on pancreatic vagal afferent discharge prior to (open bars), and after (filled bars) bilateral cervical vagotomy. Phenylbiguanide (1–10 µg/kg) before vagotomy produced significant increases in pancreatic vagal afferent discharge compared to saline, ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001. Phenylbiguanide (1–10 µg/kg) after vagotomy produced significant increases in pancreatic vagal afferent discharge compared to saline, ## P b 0.01, ###P b 0.001. Mean responses to phenylbiguanide before and after vagotomy are not significantly different (P N 0.05). All values are mean ± S.E.M., n = 5.
Fig. 7. Effects of the cholecystokinin CCK1 receptor antagonist devazepide on the excitatory responses of pancreatic vagal afferent discharge to cholecystokinin and phenylbiguanide. A, Open and filled bars represent the responses to cholecystokinin (0.1–10 µg/kg, i.v.) prior to, and after devazepide (0.5 mg/kg, i.v.), respectively. B, Open and filled bars represent the responses to phenylbiguanide (1–10 µg/kg, i.v) prior to, and after devazepide (0.5 mg/kg, i.v.), respectively. All values are mean ± S.E.M. (n = 5). ⁎⁎P b 0.01 or ⁎⁎⁎P b 0.001 compared to pre-devazepide responses.
B.M. Mussa et al. / European Journal of Pharmacology 601 (2008) 198–206
203
discharge (n = 5, P b 0.05) (Fig. 6A). As shown in Fig. 6B, lorglumide (10 mg/kg, i.v.) produced a significant inhibition of the pancreatic vagal afferent discharge responses to the 1 µg/kg dose of phenylbiguanide (n = 5, P b 0.05) without affecting the response to the highest dose of phenylbiguanide (10 µg/kg). The excitatory effects of cholecystokinin (0.1–10 µg/kg) on pancreatic vagal afferent discharge were significantly inhibited after devazepide (0.5 mg/kg, i.v.) (n = 5, P b 0.05) (Fig. 7A). Devazepide (0.5 mg/kg, i.v.) treatment also produced a significant reduction in the phenylbiguanide (1 µg/kg)induced increases in pancreatic vagal afferent discharge (n = 5, P b 0.05) (Fig. 7B). 3.4. Effects of serotonin 5-HT3 receptor blockade on pancreatic vagal afferent discharge responses to phenylbiguanide and cholecystokinin Blockade of serotonin 5-HT 3 receptors with granisetron (10 mg/kg, i.v.) significantly attenuated the phenylbiguanide (10 µg/kg)-evoked excitation in pancreatic vagal afferent discharge (n = 5, P b 0.05) with no significant effect on the responses to the lower dose (1 µg/kg) (Fig. 8A). Granisetron (10 mg/kg, i.v.) did not alter the responses to cholecystokinin (0.1–10 µg/kg) (P N 0.05; Fig. 8B). The effects of the serotonin 5-HT3 receptor antagonist, MDL 72222, on phenylbiguanide- and cholecystokinin-induced responses in pancreatic vagal afferent discharge were also tested. MDL 72222 (0.1 mg/kg, i.v.) completely abolished the excitatory responses of pancreatic vagal afferent discharge to phenylbiguanide (1–10 µg/kg) (n = 5, P b 0.05) (Fig. 9A). Compared to granisetron, MDL 72222 was more effective in reducing the phenylbiguanide-induced excitatory effects on pancreatic vagal afferent discharge. However, it did not
Fig. 9. Effects of the serotonin 5-HT3 receptor antagonist MDL 72222 on the excitatory responses of pancreatic vagal afferent discharge to phenylbiguanide and cholecystokinin. A, Open and filled bars represent the responses to phenylbiguanide (1–10 µg/kg, i.v.) prior to, and after MDL 72222 (0.1 mg/kg, i.v.), respectively. B, Open and filled bars represent the responses to cholecystokinin sulphated octapeptide (0.1–10 µg/kg, i.v.) prior to, and after MDL 72222 (0.1 mg kg− 1, i.v.), respectively. All values are mean± S.E.M. (n = 5). ⁎⁎⁎P b 0.001 compared to pre-MDL 72222 responses.
affect the cholecystokinin-induced increase in pancreatic vagal afferent discharge (n = 5, P N 0.05) (Fig. 9B). In a separate group of experiments, the effects of pre-administration of cholecystokinin (10 µg/kg) on pancreatic vagal afferent discharge-responses to phenylbiguanide were tested. Phenylbiguanide (0.01 µg/kg) produced a significant increase in pancreatic vagal afferent discharge when administered ~ 5–8 min subsequent to cholecystokinin (10 µg/kg) administration. In contrast, the same dose of phenylbiguanide produced no increase in pancreatic vagal afferent discharge following saline administration (Figs. 10 and 11). As shown in Fig. 12, these excitatory effects of phenylbiguanide (0.01 µg/ kg) on pancreatic vagal afferent discharge were significantly reduced after administration of cholecystokinin CCK1 receptor antagonists (lorglumide and devazepide).
Fig. 8. Effects of the serotonin 5-HT3 receptor antagonist granisetron on the excitatory responses of pancreatic vagal afferent discharge to phenylbiguanide and cholecystokinin. A, Open and filled bars represent the responses to phenylbiguanide (1–10 µg/kg, i.v.) prior to, and after granisetron (1 mg /kg, i.v.), respectively. B, Open and filled bars represent the responses to cholecystokinin (0.1–10 µg/kg, i.v.) prior to, and after granisetron (1 mg/kg, i.v.), respectively. All values are mean ± S.E.M (n = 5). ⁎⁎⁎P b 0.001 compared to pregranisetron responses.
Fig. 10. Effects of pre-administration of cholecystokinin on pancreatic responses to phenylbiguanide. Open and filled bars represent the responses to phenylbiguanide (0.01 µg/kg, i.v.) prior to, and after administration of cholecystokinin (10 µg/kg, i.v.), respectively. All values are mean ± S.E.M. (n = 5). ⁎⁎P b 0.01.
204
B.M. Mussa et al. / European Journal of Pharmacology 601 (2008) 198–206
Fig. 11. Effects of cholecystokinin on pancreatic vagal afferent discharge-responses to phenylbiguanide. A, Effects of phenylbiguanide (0.01 µg/kg, i.v.) on mean arterial blood pressure, heart rate and pancreatic vagal afferent discharge after administration of cholecystokinin (10 µg/kg, i.v.). The two top traces in each panel represent the normalized (in arbitrary units) and raw (in mV) discharge of the pancreatic nerve. B, Effects of phenylbiguanide (0.1 µg/kg, i.v.) on mean arterial blood pressure, heart rate and pancreatic vagal afferent discharge after administration of saline.
4. Discussion
Fig. 12. Effects of the cholecystokinin CCK1 receptor antagonists lorglumide and devazepide on the excitatory responses of pancreatic vagal afferent discharge to phenylbiguanide after administration of cholecystokinin. Open and filled bars represent effects of phenylbiguanide (0.01 µg/kg, i.v.) on pancreatic vagal afferent discharge prior to and after cholecystokinin CCK1 receptor antagonists (devazepide, 0.5 mg/kg, i.v. and lorglumide, 10 mg/kg, i.v). All values are mean ± S.E.M. (n = 5). ⁎⁎⁎P b 0.001.
Peripheral vagal afferents sense a broad range of chemical, physical and hormonal signals (Davison and Clarke, 1988; Marik and Code, 1975; Schwartz and Moran, 1998; Schwartz et al., 1995). Cholecystokinin is released from the enteroendocrine cells of the duodenum and mediates various digestive functions via activation of its receptors on peripheral vagal afferents (Adler et al., 1991; Blackshaw and Grundy, 1990; Li and Owyang, 1994; Simasko and Ritter, 2003; Viard et al., 2007). In agreement with a previous report, the results of the present study have demonstrated that pancreatic vagal afferents are highly sensitive to cholecystokinin (Niijima, 1981). Compared to Niijima's study, cholecystokinin produced more rapid and larger increases in pancreatic vagal afferent discharge and this could be due to several differences between the two studies including: (i) cholecystokinin dose range, (ii) route of cholecystokinin administration and (iii) the branch of the pancreatic vagus nerve that was used to record the afferent discharge. In the present study, intravenous administration of
B.M. Mussa et al. / European Journal of Pharmacology 601 (2008) 198–206
cholecystokinin doses in random order increased pancreatic vagal afferent discharge significantly in a dose-related manner. The responses to cholecystokinin did not change after cervical vagotomy indicating that the pancreatic vagal nerve discharge was afferent in nature. It has generally been assumed that cholecystokinin influences pancreatic secretory function via a vago-vagal reflex (Li and Owyang, 1993, 1994). Since the site of action of cholecystokinin on medullary presympathetic vasomotor neurons is on sub-diaphragmatic vagal afferents, it is conceivable that pancreatic vagal afferents are also involved in this response (Verberne and Sartor, 2004). Similarly, the depressor response to cholecystokinin is believed to be due to an inhibitory action of cholecystokinin on sympathetic vasomotor outflow (Sartor and Verberne, 2003, 2002). An important finding of this study is that the cholecystokinin CCK1 receptor mediates cholecystokinin-induced activation of pancreatic vagal afferents. This was demonstrated by the reduction in the excitatory effects of cholecystokinin on pancreatic vagal afferent discharge by pre-treatment with lorglumide or devazepide. On the other hand, blockade of serotonin 5-HT3 receptors produced no change in the cholecystokinin-evoked excitatory effects on pancreatic vagal afferent discharge suggesting that the effects of cholecystokinin are not dependent on serotonin 5-HT3 receptors. 5-HT is an important modulator of pancreatic secretion that exerts its effects via activation of duodenal mucosal vagal afferents (Li et al., 2000). The results of the present study suggest that pancreatic vagal afferents are another peripheral site at which serotonin 5-HT3 receptor agonists may act. The effects of four doses (0.01–10 µg/kg) of phenylbiguanide on pancreatic vagal afferent discharge were examined but only two doses (1 & 10 µg/kg) have produced noticeable effects. This study provides direct evidence for the involvement of serotonin 5-HT3 receptors in the effects of 5-HT on pancreatic vagal afferent discharge. Blockade of serotonin 5-HT3 receptors with two different antagonists, granisetron and MDL 72222, significantly reduced the pancreatic vagal afferent discharge response to the highest dose of phenylbiguanide (10 µg/kg). In contrast, the 1 µg/kg dose of phenylbiguanide was slightly reduced after granisetron but significantly attenuated after MDL72222. The latter seems to be more effective in inhibiting serotonin 5-HT3 receptor agonist effects on pancreatic vagal afferent discharge. Interestingly, cholecystokinin CCK1 receptor blockade with either lorglumide or devazepide significantly attenuated the responses to the low (1 µg/kg) but not the high (10 µg/kg) dose of phenylbiguanide. This novel finding suggests that phenylbiguanide acts, in part, via an interaction with cholecystokinin CCK1 receptors. Additional experiments have validated this hypothesis by showing that cholecystokinin CCK1 receptors antagonists were able to block the excitatory effects of phenylbiguanide (0.01 µg/kg) on pancreatic vagal afferent discharge which were probably due to an interaction between cholecystokinin CCK1 and serotonin 5-HT3 receptors. A similar interaction was observed in a study which demonstrated that blockade of cholecystokinin CCK1 receptors reduced the 5-HT-induced contractile responses of the guinea-pig ileum (Fortuño et al., 1999). In contrast, previous reports have shown that serotonin 5-HT3 receptors contribute to some of the effects of cholecystokinin including satiety, excitatory effects on vagal afferent neurons recorded from the nodose ganglion and the inhibitory actions on presympathetic vasomotor neurons of the rostral ventrolateral medulla (Aja, 2006; Hayes et al., 2004; Li et al., 2004; Saita and Verberne, 2003). Given the results of the present study, it is possible that some of the actions of serotonin 5-HT3 receptor antagonists are centrally-mediated. In conclusion, the present findings demonstrate that cholecystokinin and phenylbiguanide are both potent activators of pancreatic vagal afferents. Additionally, we have confirmed that cholecystokinin CCK1 and serotonin 5-HT3 receptors are mediators of the excitatory effects of cholecystokinin and 5-HT, respectively, on pancreatic vagal
205
afferent discharge. These receptors interact in a fashion that may influence pancreatic sensory function. The presence of cholecystokinin CCK1 and serotonin 5-HT3 receptors in this reflex pathway may be important for control of pancreatic exocrine and endocrine secretion. Conflict of Interest The authors state no conflict of interest. Acknowledgement This work was supported by the Austin Hospital Medical Research Foundation. References Adler, G., Beglinger, C., Braun, U., Reinshagen, M., Koop, I., Schafmayer, A., Rovati, L., Arnold, R., 1991. Interaction of the cholinergic system and cholecystokinin in the regulation of endogenous and exogenous stimulation of pancreatic secretion in humans. Gastroenterology 100, 537–543. Aja, S., 2006. Serotonin-3 receptors in gastric mechanisms of cholecystokinin-induced satiety. Am. J. Physiol. 291, R112–R114. Blackshaw, L.A., Grundy, D., 1990. Effects of cholecystokinin (cholecystokinin sulphated octapeptide-8) on two classes of gastroduodenal vagal afferent fibre. J. Auton. Nerv. Syst. 31, 191–201. Chang, T.M., Chey, W.Y., 2001. Neurohormonal control of exocrine pancreas. Curr. Opin. Gastroenterol. 17, 416–425. Chey, W.Y., Chang, T., 2001. Neural hormonal regulation of exocrine pancreatic secretion. Pancreatology 1, 320–335. Davison, J.S., Clarke, G.D., 1988. Mechanical properties and sensitivity to cholecystokinin sulphated octapeptide of vagal gastric slowly adapting mechanoreceptors. Am. J. Physiol. 255, G55–61. Fortuño, A., Ballaz, S., Del Río, J., Barber, A., 1999. Cholecystokinin sulphated octapeptide-mediated response in the activation of 5-HT receptor types in the guinea-pig ileum. J. Physiol. Biochem. 55, 85–92. Hayes, M.R., Moore, R.L., Shah, S.M., Covasa, M., 2004. 5-HT3 receptors participate in cholecystokinin sulphated octapeptide-induced suppression of food intake by delaying gastric emptying. Am. J. Physiol. 287, R817–R823. Konturek, S.J., Tasler, J., Obtulowicz, W., 1979. Effect of atropine on pancreatic responses endogenous and exogenous cholecystokinin. Am. J. Dig. Dis. 17, 911–917. Li, Y., Owyang, C., 1993. Vagal afferent pathway mediates physiological action of cholecystokinin on pancreatic enzyme secretion. J. Clin. Invest. 92, 418–424. Li, Y., Owyang, C., 1994. Endogenous cholecystokinin stimulates pancreatic enzyme secretion via vagal afferent pathway in rats. Gastroenterology 107, 525–531. Li, Y., Owyang, C., 1996. Pancreatic secretion evoked by cholecystokinin and noncholecystokinin-dependent duodenal stimuli via vagal afferent fibres in the rat. J. Physiol. (London) 494, 773–782. Li, Y., Hao, Y., Owyang, C., 1997. High-affinity cholecystokinin sulphated octapeptide-A receptors on the vagus nerve mediate cholecystokinin sulphated octapeptidestimulated pancreatic secretion in rats. Am. J. Physiol. 273, G679–G685. Li, P., Chang, T.M., Chey, W.Y., 1998. Secretin inhibits gastric acid secretion via a vagal afferent pathway in rats. Am. J. Physiol. 275, G22–G28. Li, Y., Zhu, J., Owyang, C.,1999. Electrical physiological evidence for high and low-affinity vagal cholecystokinin sulphated octapeptide-A receptors. Am. J. Physiol. 277, R469–R477. Li, Y., Hao, Y., Zhu, J., Owyang, C., 2000. Serotonin released from intestinal enterochromaffin cells mediates luminal non-cholecystokinin-stimulated pancreatic secretion in rats. Gastroenterology 118, 1197–1207. Li, J.P., Chang, T.M., Chey, W.Y., 2001a. Roles of 5-HT receptors in the release and action of secretin on pancreatic secretion in rats. Am. J. Physiol. 280, G523–G538. Li, Y., Wu, X.Y., Zhu, J.X., Owyang, C., 2001b. Intestinal serotonin acts as paracrine substance to mediate pancreatic secretion stimulated by luminal factors. Am. J. Physiol. 281, G916–G923. Li, Y., Wu, X.Y., Owyang, C., 2004. Serotonin and cholecystokinin synergistically stimulate rat vagal primary afferent neurones. J. Physiol. 55, 651–662. Li, Y., Wu, X., Yao, H., Owyang, C., 2005. Secretin activates vagal primary afferent neurons in the rat: evidence from electrophysiological and immunohistochemical studies. Am. J. Physiol. 289, G745–G752. Liao, Z., Li, Z.S., Lu, Y., Wang, W.Z., 2005. Glutamate receptors within the nucleus of solitary tract contribute to pancreatic secretion stimulated by intraduodenal hypertonic saline. Auton. Neurosci. 120, 62–67. Love, J.A., Yi, E., Smith, T.G., 2006. Autonomic pathways regulating pancreatic exocrine secretion. Auton. Neurosci. 16, 1–16. Marik, F., Code, C.F., 1975. Control of the interdigestive myoelectric activity in dogs by the vagus nerves and pentagastrin. Gastroenterology 69, 387–395. Moran, T.H., Smith, G.P., Hostetler, A.M., McHugh, P.R., 1987. Transport of cholecystokinin (cholecystokinin sulphated octapeptide) binding sites in subdiaphragmatic vagal branches. Brain Res. 415, 149–152. Moriarty, P., Dimaline, R., Thompson, D.G., Dockray, G.J., 1997. Characterization of cholecystokininA and cholecystokininB receptors expressed by vagal afferent neurons. Neuroscience 79, 905–913. Mussa, B., Verberne, A.J., 2008. Activation of the dorsal vagal nucleus increases pancreatic exocrine secretion in the rat. Neurosci. Lett. 433, 71–76.
206
B.M. Mussa et al. / European Journal of Pharmacology 601 (2008) 198–206
Niebergall-Roth, E., Singer, M.V., 2001. Central and peripheral neural control of pancreatic exocrine secretion. J. Physiol. Pharmacol. 52, 523–538. Niijima, A., 1981. Visceral afferents and metabolic function. Diabetologia 20, 325–330. Owyang, C., 1996. Physiological mechanisms of cholecystokinin action on pancreatic secretion. Am. J. Pysiol. 34, G1–G7. Owyang, C., Logsdon, C.D., 2004. New insights into neurohormonal regulation of pancreatic secretion. Gastroenterology 127, 957–969. Raybould, H.E., Glatzle, J., Robin, C., Meyer, J.H., Phan, T., Wong, H., Sternini, C., 2003. Expression of 5-HT3 receptors by extrinsic duodenal afferents contribute to intestinal inhibition of gastric emptying. Am. J. Physiol. 284, G367–G372. Saita, M., Verberne, A.J., 2003. Roles for cholecystokinin sulphated octapeptide1 and 5-HT3 receptors in the effects of cholecystokinin sulphated octapeptide on presympathetic vasomotor neuronal discharge in the rat. Br. J. Pharmacol. 139, 415–423. Sartor, D.M., Verberne, J.M.J., 2002. Cholecystokinin selectively affects presympathetic vasomotor neurons and sympathetic vasomotor outflow. Am. J. Physiol. 280, R1174–R1184. Sartor, D.M., Verberne, A.J.M., 2003. Phenotypic identification of rat rostroventrolateral medullary presympathetic vasomotor neurons inhibited by exogenous cholecystokinin. J. Comp. Neurol. 456, 467–479. Schwartz, G.J., Moran, T.H., 1998. Duodenal nutrient exposure elicits nutrient-specific gut motility and vagal afferent signals in rat. Am. J. Physiol. 274, R1236–R1242. Schwartz, G.J., Tougas, G., Moran, T.H., 1995. Integration of vagal afferent responses to duodenal loads and exogenous cholecystokinin sulphated octapeptide in rats. Peptides 16, 707–711.
Simasko, M.S., Ritter, C.R., 2003. Cholecystokinin activates both A- and C-type vagal afferent neurons. Am. J. Physiol. 285, G1204–G1213. Soudah, H.C., Lu, Y., Hasler, W.L., Owyang, C., 1992. Cholecystokinin at physiological levels evokes pancreatic enzyme secretion via a cholinergic pathway. Am. J. Physiol. 263, G102–G107. Travagli, R.A., Gillis, R.A., Rossiter, C.D., Vicini, S., 1991. Glutamate and GABA-mediated synaptic currents in neurons of the rat dorsal motor nucleus of the vagus. Am. J. Physiol.: Gastrointest. Liver Physiol. 260. Travagli, R.A., Hermann, G.E., Browning, K.N., Rogers, R.C., 2006. Brainstem circuits regulating gastric function. Annu. Rev. Physiol. 68, 279–305. Verberne, A.J.M., Sartor, D.M., 2004. Cholecystokinin sulphated octapeptide-induced inhibition of presympathetic vasomotor neurons: dependence on subdiaphragmatic vagal afferents and central NMDA receptors in the rat. Am. J. Physiol., Regul. Integr. Comp. Physiol. 287, R809–R816. Viard, E., Zheng, Z., Wan, S., Travagli, R.A., 2007. Vagally mediated, nonparacrine effects of cholecystokinin-8 s on rat pancreatic exocrine secretion. Am. J. Physiol. 293, G493–G500. Zarbin, M.A., Wamsley, J.K., Innis, R.B., Kuhar, M.J., 1981. Cholecystokinin receptors: presence and axonal flow in the rat vagus nerve. Life. Sci. 29, 697–705.