Marcel Miampamba

Activation of Corticotropin-Releasing Factor Receptor 2 Mediates the Colonic Motor Coping Response to Acute Stress in Rodents

BACKGROUND & AIMS Corticotropin-releasing factor receptor-1 (CRF(1)) mediates the stress-induced colonic motor activity. Less is known about the role of CRF(2) in the colonic response to stress. METHODS We studied colonic contractile activity in rats and CRF(2)-/-, CRF-overexpressing, and wild-type mice using still manometry; we analyzed defecation induced by acute partial-restraint stress (PRS), and/or intraperitoneal injection of CRF ligands. In rats, we monitored activation of the colonic longitudinal muscle myenteric plexus (LMMP) neurons and localization of CRF(1) and CRF(2) using immunohistochemical and immunoblot analyses. We measured phosphorylation of extracellular signal-regulated kinase 1/2 by CRF ligands in primary cultures of LMMP neurons (PC-LMMPn) and cyclic adenosine monophosphate (cAMP) production in human embryonic kidney-293 cells transfected with CRF(1) and/or CRF(2). RESULTS In rats, a selective agonist of CRF(2) (urocortin 2) reduced CRF-induced defecation (>50%), colonic contractile activity, and Fos expression in the colonic LMMP. A selective antagonist of CRF(2) (astressin(2)-B) increased these responses. Urocortin 2 reduced PRS-induced colonic contractile activity in wild-type and CRF-overexpressing mice, whereas disruption of CRF(2) increased PRS-induced colonic contractile activity and CRF-induced defecation. CRF(2) colocalized with CRF(1) and neuronal nitric oxide synthase in the rat colon, LMMP, and PC-LMMPn. CRF-induced phosphorylation of extracellular signal-regulated kinase in PC-LMMPn; this was inhibited or increased by a selective antagonist of CRF(1) (NBI35965) or astressin(2)-B, respectively. The half maximal effective concentration, EC(50), for the CRF-induced cAMP response was 8.6 nmol/L in human embryonic kidney-293 cells that express only CRF(1); this response was suppressed 10-fold in cells that express CRF(1) and CRF(2). CONCLUSIONS In colon tissues of rodents, CRF(2) activation inhibits CRF(1) signaling in myenteric neurons and the stress-induced colonic motor responses. Disruption of CRF(2) function impairs colonic coping responses to stress.

Expression of pituitary adenylate cyclase-activating polypeptide and PACAP type 1 receptor in the rat gastric and colonic myenteric neurons.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is known to regulate gastric acid secretion and intestinal motility. In the present study, the pattern of distribution of PACAP and PACAP type 1 receptor (PAC1) immunoreactivities were examined in the rat stomach and distal colon using a specific polyclonal antibody raised against rat/human PAC1. Western blot of the membrane preparations of NIH/3T3 cells transfected with the human PAC1 obtained by using rabbit polyclonal anti-PAC1 antibody showed a protein band with a molecular mass of approximately 50 kDa. NIH/3T3 cells transfected with the human PAC1 and incubated with the anti-PAC1 antibody displayed surface cell-type immunoreactivity, which was internalized following ligand exposure. In gastric or colonic longitudinal muscle/myenteric plexus (LMMP) whole mount preparations as well as cryostat sections, PACAP immunoreactivity was observed in cell bodies within the myenteric ganglia and nerve fibers in the muscle layers and mucosa. PAC1 immunoreactivity was confined mainly on the surface of the nerve cells. PACAP and PAC1 immunoreactivities showed a similar pattern of distribution in gastric and colonic tissues. Adjacent sections or LMMP whole mount preparations labeled with protein gene product 9.5 (PGP 9.5) revealed the neuronal identity of myenteric cells bearing PAC1. The neuronal localization of PACAP and PAC1 receptors supports their role in the neural regulation of gastric acid secretion and gastrointestinal motor function.

Role of brainstem TRH/TRH-R1 receptors in the vagal gastric cholinergic response to various stimuli including sham-feeding

Pavlovs pioneering work established that sham-feeding induced by sight or smell of food or feeding in dogs with permanent esophagostomy stimulates gastric acid secretion through vagal pathways. Brain circuitries and transmitters involved in the central vagal regulation of gastric function have recently been unraveled. Neurons in the dorsal vagal complex including the dorsal motor nucleus of the vagus (DMN) express thyrotropin-releasing hormone (TRH) receptor and are innervated by TRH fibers originating from TRH synthesizing neurons in the raphe pallidus, raphe obscurus and the parapyramidal regions. TRH injected into the DMN or cisterna magna increases the firing of DMN neurons and gastric vagal efferent discharge, activates cholinergic neurons in gastric submucosal and myenteric plexuses, and induces a vagal-dependent, atropine-sensitive stimulation of gastric secretory (acid, pepsin) and motor functions. TRH antibody or TRH-R1 receptor oligodeoxynucleotide antisense pretreatment in the cisterna magna or DMN abolished vagal-dependent gastric secretory and motor responses to sham-feeding, 2-deoxy-D-glucose, cold exposure and chemical activation of cell bodies in medullary raphe nuclei. TRH excitatory action in the DMN is potentiated by co-released prepro-TRH-(160-169) flanking peptide, Ps4 and 5-HT, and inhibited by a number of peptides involved in the stress/immune response and inhibition of food-intake. These neuroanatomical, electrophysiological and neuropharmacological data are consistent with a physiological role of brainstem TRH in the central vagal stimulation of gastric myenteric cholinergic neurons in response to several vagal dependent stimuli including sham-feeding.

Water avoidance stress activates colonic myenteric neurons in female rats.

Stress stimulates colonic motor function and plays a role in functional bowel disorders, prevalently in women. We examined, in conscious female rats, the influence of water avoidance stress for 60 min on colonic myenteric neuron activity using immunohistochemical detection of Fos as a marker of neuronal activity. In control rats, Fos immunoreactive nuclei were rare in proximal and distal colon and no defecation was observed. Water avoidance stimulated fecal pellet output, which was associated with Fos expression in myenteric ganglia of proximal and distal colon including in a population of peripheral choline acetyltransferase-immunoreactive neurons. Atropine blocked fecal pellet output but not Fos expression in myenteric ganglia. These results indicate that psychological stress stimulates the activity of colonic cholinergic myenteric neurons.

Brain-gut interactions between central vagal activation and abdominal surgery to influence gastric myenteric ganglia Fos expression in rats.

We previously showed that medullary thyrotropin-releasing hormone (TRH) or the stable TRH agonist, RX-77368 administered intracisternally induces vagal-dependent activation of gastric myenteric neurons and prevents post surgery-induced delayed gastric emptying in rats. We investigated whether abdominal surgery alters intracisternal (ic) RX-77368 (50 ng)-induced gastric myenteric neuron activation. Under 10 min enflurane anesthesia, rats underwent an ic injection of saline or RX-77368 followed by a laparotomy and a 1-min cecal palpation, or no surgery and were euthanized 90 min later. Longitudinal muscle/myenteric plexus whole-mount preparations of gastric corpus and antrum were processed for immunohistochemical detection of Fos alone or double labeled with protein gene-product 9.5 (PGP 9.5) and vesicular acetylcholine transporter (VAChT). In the non surgery groups, ic RX-77368 induced a 17 fold increase in Fos-expression in both gastric antrum and corpus myenteric neurons compared to saline injected rats. PGP 9.5 ascertained the neuronal identity of myenteric cells expressing Fos. In the abdominal surgery groups, ic RX-77368 induced a significant increase in Fos-expression in both the corpus and antrum myenteric ganglia compared with ic saline injected rats which has no Fos in the gastric myenteric ganglia. However, the response was reduced by 73-78% compared with that induced by ic RX 77368 without surgery. Abundant VAChT positive nerve fibers were present around Fos positive neurons. These results indicate a bidirectional interaction between central vagal stimulation of gastric myenteric neurons and abdominal surgery. The modulation of gastric vagus-myenteric neuron activity could play an important role in the recovery phase of postoperative gastric ileus.