An improved method of evaluation of drug-evoked changes in gastric emptying in mice.

INTRODUCTION: The increased availability of transgenic mice prompts a need for the adaptation to mice of whole-animal assays traditionally performed in larger laboratory animals. Gastric emptying studies are frequently conducted in dogs and rats. Mouse-based gastric emptying models currently available often use inert, nonnutrient liquid meals containing nonabsorbable markers or radionuclides. We have developed a mouse gastric emptying assay that features a favorable throughput and the use of a semisolid, high-calorie meal. METHODS: A carbohydrate- and protein-rich semisolid test meal was prepared from common laboratory reagents. Gastric emptying was determined by subtracting the mass of test meal remaining in the stomach from the mass of test meal administered. A time-course study of basal emptying of a semisolid, paste-like test meal high in carbohydrate and protein from the stomachs of overnight-fasted mice was conducted. Agents known to either inhibit (propantheline, 0.3-10 mg/kg sc; corticotropin-releasing factor [CRF], 3-100 nmol/kg ip) or accelerate (metoclopramide, 1-10 mg/kg ip; bethanechol, 1-30 mg/kg ip) gastric emptying were tested. A single time-point variation of the assay can be used for quickly screening compounds for effects on gastric emptying. RESULTS: In time-course studies, the test meal emptied from the stomach with a half-emptying time of 30.6 min (95% CI: 27.3-34.7). The gastric emptying data were successfully modeled by a two-parameter exponential decay function. No lag phase was observed, indicating that the meal empties from the stomach as a liquid. The anticholinergic agent propantheline increased gastric half-emptying time (t(1/2)) approximately threefold, while metoclopramide decreased gastric half-emptying time approximately twofold compared to basal emptying. Single time-point screening studies correctly detected the gastrokinetic activity of bethanechol and the inhibitory effect of CRF. DISCUSSION: The mouse gastric emptying assay reported here is simple, inexpensive, and not labor-intensive. It is capable of detecting either stimulation or inhibition of gastric motor activity. This assay should prove useful for identifying drug-evoked changes in gastric emptying as well as for assessing the gastric motility effects of altered gene expression in genetically modified mice.

Orphanin FQ/nociceptin: a novel neuromodulator of gastrointestinal function?

Orphanin FQ/nociceptin (OFQ/N) has been immunohistochemically localized in the rat enteric nervous system, and mRNA signals for its precursor (i.e. prepro-OFQ/-N) and cognate receptor ORL-1 are expressed in the intestinal tract. OFQ/N inhibits neurogenic contractions in a variety of stomach and small intestine preparations in vitro, but contracts rodent colon. In vivo, it acts at peripheral and central nervous system sites to stimulate or inhibit mechanical activity in the stomach and colon, respectively. Thus, OFQ/N acts as a neuromodulator of gastrointestinal motility and may have additional roles in regulation of intestinal blood flow, active ion transport, and immunity.

Expression of nociceptin/OFQ receptor and prepro-nociceptin/OFQ in lymphoid tissues.

This study was undertaken to examine the presence of functional nociceptin/orphanin FQ (N/OFQ) receptors in the immune system. Receptor mRNA signals were detected by RT-PCR in porcine thymus, lymph nodes, spleen and freshly-isolated splenocytes; the distribution of prepro-nociceptin/-orphanin FQ (PP-N/-OFQ) mRNA was similar, with the exception of lymph nodes. However, specific [(3)H]nociceptin binding sites were not detected in rat or porcine lymphoid tissues, and 0.1-100 nM nociceptin had no effect on forskolin-stimulated cyclic AMP concentrations in porcine splenocytes. Thus, it appears that nociceptin/orphanin FQ receptor mRNA, but not a functional receptor protein is expressed in the immune system.

Cloning, expression and functional role of a nociceptin/orphanin FQ receptor in the porcine gastrointestinal tract.

The heptadecapeptide nociceptin/orphanin FQ is the cognate ligand for the opioid receptor-like orphanin FQ (OFQ) receptor, a member of the G protein-coupled receptor superfamily. The gastrointestinal tract is a major site of opioid action, and preliminary evidence suggests that an OFQ receptor may be expressed in rat small intestine. We addressed the hypothesis that this receptor is expressed in the gastrointestinal tract of the pig, a model for the human digestive system. A 1205-bp cDNA was isolated from porcine forebrain which contained the 370 amino acid open reading frame encoding the OFQ receptor. The receptor mRNA is likely to arise from a single gene, as determined by Southern blotting of porcine genomic DNA restriction digests using a porcine OFQ receptor cDNA probe. A semi-nested reverse transcriptase-polymerase chain reaction survey of receptor mRNA indicates that it is expressed in the porcine cerebral cortex and kidney, and along the length of the gastrointestinal tract. OFQ decreased initial contractile responses of porcine ileal smooth muscle strips to trains of electrical field stimulation with an IC50 value of 1.3 nM; its effects were resistant to the opioid antagonist, naloxone. The peptide, at concentrations > or =3 nM, also attenuated Isc elevations evoked by electrical transmural stimulation of mucosa-submucosa sheets from porcine ileum. The actions of OFQ appeared to differ from those previously reported for opioid receptor agonists in these tissue preparations. These results indicate that an OFQ receptor is expressed in the porcine intestine which modulates the neural control of intestinal smooth muscle contractility and mucosal transport.

Peripheral and central actions of orphanin FQ (nociceptin) on murine colon.

Orphanin FQ (OFQ), also known as nociceptin, is a recently isolated endogenous peptide with a structure similar to the endogenous opioid peptides. The present study examines the actions of centrally administered OFQ on in vivo murine gastrointestinal and colonic transit as well as the actions of OFQ on the isolated colon. Intracerebroventricular injections of OFQ dose dependently inhibited colonic propulsive activity. OFQ inhibition of colonic propulsion was unaffected by coadministration of the competitive opioid receptor antagonist naltrexone. A subadditive response was observed when approximately equipotent doses of either morphine sulfate or the delta-agonist DPDPE were coadministered with OFQ. No subadditivity was observed with coadministration of the micro-agonist DAMGO, suggesting a functional interaction between OFQ and delta-opioid central pathways regulating colonic transit. High, but not low, doses of OFQ also inhibited the transit of a nonabsorbable charcoal marker through the stomach and/or small intestine. OFQ potently contracted isolated colon preparations; contractile activity was abolished by TTX or chlorpromazine. Our results suggest that OFQ may be an important peptide ligand acting on a novel inhibitory neural pathway that modulates gastrointestinal transit.

The porcine mu opioid receptor: molecular cloning and mRNA distribution in lymphoid tissues.

The porcine mu opioid receptor (pMOR), was cloned from cerebral cortex RNA using PCR methodologies. Porcine MOR is 96% identical with human MOR in amino acid sequence. An RT-PCR survey for pMOR mRNA indicated that pMOR is widely distributed in the gut, and is present in thymus and Peyer's patches but absent in other immune tissues and in isolated immune cells. Based on these findings, it appears that opioids do not exert an immunosuppressive effect through direct interaction with the mu-opioid receptor on immune cells. In certain tissues, however, opioids may modulate immune function indirectly through neuronal MOR.

Delta-opioid receptor mRNA expression and immunohistochemical localization in porcine ileum.

Opiates have potent antidiarrheal actions that are mediated in part by delta-opioid receptors (DOR). We examined DOR localization within subregions of porcine ileum, a tissue analogous to human small bowel. A partial cDNA sequence for porcine DOR was obtained after reverse transcription-polymerase chain reaction cloning of forebrain RNA; it encoded the end of transmembrane domain 1 through the beginning of transmembrane domain 7 and exhibited 93% nucleotide identity with human DOR. Positive signals for DOR mRNA were found in all subregions of the porcine ileal wall. With an antiserum recognizing an N-terminal epitope in murine DOR, DOR-like immunoreactivity was localized in neurons within myenteric and submucous ganglia, longitudinal and circular smooth muscle, and villous lamina propria. The DOR agonist [D-Ser2, Leu5, Thr6]enkephalin (DSLET) attenuated circular smooth muscle contractions in porcine ileum that were evoked by electrical stimulation of myenteric cholinergic neurons. These results are consistent with previous reports of the DOR-mediated neuromodulation that underlies the antipropulsive and antisecretory effects of opioids in the intestinal tract.

GDNF is abundant in the adult rat gut.

Glial derived neurotrophic factor (GDNF) is essential for the development of the enteric nervous system (ENS). Although previous work has measured GDNF mRNA levels, little is known about the concentration of GDNF protein produced in developing or adult tissues. The aim of this study was to quantitate the concentration of GDNF protein in various tissues of the developing and adult rat and in adult human gut. A two site antibody immunoassay was used to quantitate GDNF using recombinant rat GDNF as a standard. In the adult rat gastrointestinal tract the intestine contained the highest concentration of GDNF while the stomach and esophagus have the lowest concentrations. The isolated muscular wall of the intestine has approximately four times the GDNF concentration of the intact intestine. Other tissues with smooth muscle such as the aorta and urinary bladder contain moderate GDNF concentrations. In contrast, GDNF is barely detectable in the adult kidney and liver. High concentrations of GDNF were also detected in human colon and jejunum. As development proceeds in the rat, there is a tendency for the concentration of GDNF to increase in the intestine but decrease in other tissues. Treatment of the jejunum with the cationic surfactant benzyldimethyltetradecylammonium chloride (BAC) results in an increase in the number of smooth muscle cells, a decrease in myenteric neurons, and an increase in the concentration of GDNF in homogenates of intestine. The observations that GDNF concentrations are high in the adult intestine suggest that this growth factor may be important for the maintenance of the adult ENS.

Linkage assignment of eleven genes to the porcine genome.

We report comparative linkage mapping of eleven genes in the swine genome by RFLP analysis. These genes include: Acid phosphatase type 5 (ACP5), Cholecystokinin Type B Receptor (CCKBR), Antibiotic Peptide (FALL39), Insulin-like Growth Factor 1 Receptor (IGF1R), Integrin Alpha M (ITGAM), Integrin Beta 2 (ITGbeta2), Opioid Receptor Mu-1 (OPRM1), Pro-hormone Converter (PC1/3), Retinol Binding Protein 3 (RBP3), Ribosomal DNA (RNR1), and Zona Pellucida Glycoprotein 1 (ZP1). The CCKBR and ITGbeta2 loci define the ends of the linkage groups on Chromosomes (Chro) (SSC) 9p and 13qter, respectively.

Electrophysiological and pharmacological responses of chronically denervated lower esophageal sphincter of the opossum.

BACKGROUND & AIMS: Achalasia is characterized by loss of myenteric neurons and incomplete relaxation of the lower esophageal sphincter (LES). The aim of this study was to develop an achalasia model in the opossum using the surfactant benzyldimethyltetradecylammonium chloride (BAC). This study further characterizes the achalasia model. METHODS: BAC or saline was injected circumferentially into the LES of 14 adult opossums. Eight months after injection, manometry, isolated muscle bath studies, electrical field stimulation, and histochemical analysis were performed. RESULTS: Manometrically, the LES of BAC-treated opossums showed higher pressures (38.7 +/- 12 mm Hg vs. 17 +/- 3.0 mm Hg) and reduced esophageal body contraction amplitudes (4.2 +/- 3 mm Hg vs. 27.4 +/- 12 mm Hg). Isolated muscle strips challenged with carbachol and sodium nitroprusside contracted and relaxed similarly to controls. Electrical field stimulation failed to induce relaxation in BAC-treated tissue but did induce contraction. Contractile responses were markedly reduced by tetrodotoxin and atropine in BAC-treated animals and controls. An altered nitric oxide system was shown by the lack of response to L-arginine and N omega-nitro-L-arginine. Histology showed loss of myenteric neurons and increased cholinergic nerve bundles. CONCLUSIONS: Loss of NO inhibitory myenteric neurons markedly reduces the relaxation of the LES, and histology and pharmacological responses suggest a proliferation of cholinergic nerves into the LES contributing to the static elevated pressures of the amyenteric LES.

Myenteric denervation of rat jejunum alters calcium responsiveness of intestinal smooth muscle.

BACKGROUND/AIMS: Long-term myenteric and extrinsic denervation of a segment of rat jejunum results in increased stress generation by the longitudinal muscle layer of the denervated segment 15 days after denervation. This study examined whether alterations in the properties of either cell membrane calcium channels and/or sarcoplasmic reticular Ca(2+)-adenosine triphosphatase (ATPase) contribute to the increased stress development. METHODS: The effects of the calcium channel blocker nifedipine and the sarcoplasmic reticular Ca(2+)-ATPase inhibitor cyclopiazonic acid on the contractile activity of denervated and control smooth muscle were determined. RESULTS: The ability of nifedipine to inhibit KCl-induced contractions was significantly increased in denervated tissues; however, there was no difference in the potency of nifedipine when tissues were stimulated with carbachol. Calcium concentration-response curves obtained in the presence of either KCl or carbachol were determined in tissues previously depleted of calcium. Long-term denervated tissues showed an increased sensitivity to calcium and a decreased maximum contractile response after stimulation with carbachol. Cyclopiazonic acid inhibited repletion of intracellular calcium stores of control muscle but had no effect in denervated tissue. CONCLUSIONS: Long-term denervation of a segment of rat small intestine results in profound alterations in calcium metabolism at the cell membrane and, to a lesser extent, at the sarcoplasmic reticulum of smooth muscle cells of the longitudinal muscle layer.

Increased active stress generation of denervated rat intestinal smooth muscle: functional analysis of muscarinic receptor population.

The effects of denervation on the active stress production by the longitudinal muscle (LM) layer of rat jejunum were examined. Extrinsic and myenteric denervation of a segment of rat jejunum was accomplished by the serosal application of the cationic surfactant benzyldimethyltetradecylammonium chloride (BAC). Isolated muscle contraction experiments revealed that the LM of the jejunum taken from rats treated with BAC 15 days before developed significantly increased active stress in response to bethanechol and carbachol, but not in response to potassium chloride. No change in -log EC50 values of any of the agonists was observed in the denervated LM layer, although a significant increase in the slope of the carbachol and bethanechol concentration-response curves was observed in the denervated LM. Schild analysis of several muscarinic antagonists revealed a 3-fold increase in the apparent dissociation constant of the M2 antagonist methoctramine in BAC-treated LM. These results suggest that the increased responsiveness of the denervated LM may originate in the muscarinic receptor population of the myocytes.

Proliferation of mast cells in the smooth muscle of denervated rat jejunum.

Mast cell hyperplasia and changes in phenotypic characteristics subsequent to myenteric and extrinsic denervation of a segment of rat jejunum were studied. The myenteric plexus and extrinsic nerves were ablated by serosal application of the cationic surfactant benzyldimethyltetradecylammonium chloride. There was a four-fold increase in the number of mast cells in the smooth muscle layers 15 days after denervation. This increase was sustained for at least 90 days after treatment. No increase in mast cell number was observed in the villus-crypt axis of the jejunum. Berberine sulfate fluorescent detection of heparin-containing mast cells demonstrated that a change in mast cell phenotype occurred between 20 and 90 days after the denervation procedure. The fact that myeloperoxidase activity was the same in denervated and control tissue within 5 days of denervation demonstrates the lack of a chronic inflammatory reaction. Our results suggest that mast cells might play a role in the gut wall re-modeling processes.

Chronic denervation of rat jejunum results in cholinergic supersensitivity due to reduction of cholinesterase activity.

We examined the effects of chronic myenteric/extrinsic denervation of a segment of rat small intestine on specific acetylcholinesterase (AChE) and nonspecific cholinesterase (ChE) activities. Myenteric/extrinsic denervation of a 5-cm length of jejunum was accomplished by serosal application of the cationic surfactant benzyldimethyltetradecylammonium chloride. Fifteen days after denervation, biochemical determinations of AChE and ChE revealed that the smooth muscle layers, but not the mucosa, of denervated tissues have decreased levels of both AChE (approximately 65% decreased) and ChE (approximately 30% decreased). Histochemical staining of sections of denervated small intestine for AChE activity failed to detect any AChE activity in the region formerly occupied by the myenteric plexus. There appeared to be a qualitative decrease in ChE staining in the longitudinal muscle of denervated small intestine. Isolated muscle contraction experiments were conducted with the aim of determining changes in muscle responsiveness to acetylcholine (ACh) subsequent to denervation. Denervated circular and longitudinal muscle layers displayed increased sensitivity to ACh compared to nondenervated tissues (pEC50 values: 5.23 vs. 3.53, circular muscle; 5.33 vs. 4.08, longitudinal muscle). Incubation of tissues with 10 microM neostigmine caused a similar shift in pEC50 values of ACh concentration-response curves from denervated circular and longitudinal muscle layers (approximately 20% increase in pEC50). In contrast to the similar effects of neostigmine on both muscle layers of denervated tissues, neostigmine exerted a differential effect on the muscle layers of control intestine. Neostigmine produced a 65% increase in pEC50 value in control circular muscle and a 49% increase in pEC50 value in control longitudinal muscle.(ABSTRACT TRUNCATED AT 250 WORDS)