Central neuropeptide Y receptors are involved in 3rd ventricular ghrelin induced alteration of colonic transit time in conscious fed rats

BMC Gastroenterology Central neuropeptide Y receptors are involved in 3rd ventricular ghrelin induced alteration of colonic transit time in conscious fed rats Johannes J Tebbe 2 Clemens G Tebbe 2 Silke Mronga 2 Michael Ritter 1 Martin KH Schfer 0 0 Department of Molecular Neuroscience, Institute of Anatomy and Cell Biology, Philipps Universitat Marburg , 35033 Marburg , Germany 1 Department of Internal Medicine, Division Cardiology, Philipps Universitat Marburg , 35033 Marburg , Germany 2 Department of Internal Medicine, Division Gastroenterology - Endocrinology, Philipps Universitat Marburg , 35033 Marburg , Germany Background: Feeding related peptides have been shown to be additionally involved in the central autonomic control of gastrointestinal functions. Recent studies have shown that ghrelin, a stomachderived orexigenic peptide, is involved in the autonomic regulation of GI function besides feeding behavior. Pharmacological evidence indicates that ghrelin effects on food intake are mediated by neuropeptide Y in the central nervous system. Methods: In the present study we examine the role of ghrelin in the central autonomic control of GI motility using intracerobroventricular and IP microinjections in a freely moving conscious rat model. Further the hypothesis that a functional relationship between NPY and ghrelin within the CNS exists was addressed. Results: ICV injections of ghrelin (0.03 nmol, 0.3 nmol and 3.0 nmol/5 l and saline controls) decreased the colonic transit time up to 43%. IP injections of ghrelin (0.3 nmol - 3.0 nmol kg-1 BW and saline controls) decreased colonic transit time dose related. Central administration of the NPY1 receptor antagonist, BIBP-3226, prior to centrally or peripherally administration of ghrelin antagonized the ghrelin induced stimulation of colonic transit. On the contrary ICV-pretreatment with the NPY2 receptor antagonist, BIIE-0246, failed to modulate the ghrelin induced stimulation of colonic motility. Conclusion: The results suggest that ghrelin acts in the central nervous system to modulate gastrointestinal motor function utilizing NPY1 receptor dependent mechanisms. - Background The presence or absence of food in the gut stimulates the release of several regulatory peptides. These orexigenic (NPY, AGRP, ghrelin, MCH, Orexin-A, ...) and anorexigenic (CRF, CCK, CART, GLP-1, leptin, insulin, ...) peptides participating in the hypothalamic control of feeding behavior and satiety have been shown to be additionally involved in the autonomic control of gastointestinal (GI) functions like secretion and motility. For example fasted motor activity of the GI tract, e.g. the colon, is observed after intracerebroventricular (ICV) injection of neuropeptide Y whereas CRF ICV-treatment cause the disruption of fasted colonic motor activity [1]. Stomach-derived ghrelin is the first peripheral orexigenic peptide identified [2-6]. There is convincing evidence from several groups of investigators that ghrelin acts in the CNS and the periphery to simulate not only feeding but also GI function such as gastric acid secretion and gastric motility in rodents [7,9-11]. However, it is still unknown whether ghrelin is involved in the CNS control of other digestive functions besides gastric acid secretion and motility. Recent studies suggest that CNS-signaling by circulating ghrelin is mediated downstream by neurons of arcuate nucleus and the paraventricular nucleus of the hypothalamus, in particular, neurons expressing neuropeptide Y and agouti-related protein (AGRP) [12-14]. Furthermore it has been demonstrated that there is an anatomical interaction and functional relationship between ghrelin and neuropeptide Y. Using electrophysiological recordings Cowley et al have found that ghrelin stimulated the activity of arcuate NPYergic neurons and mimicked the effect of NPY in the paraventricular nucleus of the hypothalamus [15]. In addition ghrelin simulates food intake through hypothalamic NPY1 receptors [1,16,17]. Thus, the question came up "are NPY receptors involved in the ghrelin effect on GI function"? Among others, neuropeptide Y plays a role in the CNS control of gastrointestinal function [1,18]. NPY activates at least six receptor subtypes, NPY1 to NPY6. NPY binds preferentially with high affinity to Y1 and Y2 receptors, and there is evidence suggesting that these two receptor subtypes are involved in CNS regulation of digestive function by NPY action in arcuate nucleus and the paraventricular nucleus of the hypothalamus [18]. Taken together there is overwhelming evidence that ghrelin, beside its satiety modulatory capacity, is involved in the CNS control of digestive function of the upper gastrointestinal tract. In the CNS ghrelin and NPY, the most potent orexigenic neuropeptides known, are anatomical associated and functionally related. Moreover hypothalamic NPYergic neurons are downstream mediators of feeding related ghrelin action. In the present study we scrutinize the hypothesis that central neuropeptide Y receptor activation is involved in the ghrelin induced modulation of gastrointestinal motility using a microinjection-model in conscious fed and freely moving rats. tion for the protection of animals and were licensed and supervised by the appropriate government body. Male Sprague-Dawley rats with a mean body weight of 350 50 g were maintained on a 12 : 12 h photoperiod. They were housed in colony cages under conditions of controlled humidity and temperature (22 2C) for at least 7 days prior to the surgical procedure. The animals were fed a standard rat diet (Altromin, Lage, Germany) an tap water ad libitum. After surgical procedures, rats were housed individually. During experimental procedure the animals had continuous access to food and water. Drugs Ghrelin (Bachem, Heidelberg, Germany) doses of 0.03 nmol (100 ng), 0.3 nmol (1 g) and 3 nmol (10 g)/5 l were dissolved in 0.15 M sterile saline (B. Braun, Melsungen, Germany). The NPY1 receptor antagonist, BIBP-3226 (200 nmol/5 l; Sigma-RBI, Natrick, MA, USA) [see Ref.: [19]] and the NPY2 receptor antagonist, BIIE-0246 (120 nmol/5 l; Boehringer-Ingelheim, Biberach, Germany) [see Ref.: [21]] were dissolved in sterile 0.15 M saline. The NPY receptor antagonists were used in similar equipotent nanomolar concentrations. The used intracerebroventricular concentrations of the receptor antagonists were comparable with the ICV-dosages used by other groups in rodents [18,20]. Probes were aliquoted and frozen (80C). Fresh aliquots were thawed on each experimental day before injections. Any excess was discarded. In our hands nanomolar concentrations of BIBP-3226 and BIIE0246 were effective in antagonization of NPY receptor subtypes without any side effects. In particular no central depressive effects or conspicuous behavior was observed after BIBP-3226 treatment [18]. Cerebral cannulas For surgical procedures, rats were anesthetized with a mixture of ketamine (75 mg kg-1 i.p., Parke- (...truncated)