Acute stress increases colonic paracellular permeability in mice through a mast cell-independent mechanism: Involvement of pancreatic trypsin

AimsIncreased colonic paracellular permeability (CPP) is a key feature of gastro-intestinal disorders as irritable bowel syndrome and inflammatory bowel diseases. Stress stimulates exocrine pancreatic secretion through cholinergic pathways, and trypsin is known to increase CPP. Consequently we have investigated in this work whether trypsin released into the gut lumen following an acute stress may participate to the short-term increase in CPP.Main methodsMice were treated with atropine or a non-selective CRF (corticotropin-releasing factor) receptor antagonist (α-helical CRF (9–41)), before being submitted to a 2-h stress session. Then, CPP and protease activity in colonic contents (total proteolytic, trypsin activity, and mouse mast cell protease (MMCP)-1 levels) were determined. The effects of colonic contents from sham-stressed or stressed animals on CPP were evaluated in mice colonic tissues mounted in Ussing chambers, in presence or not of soybean trypsin inhibitor (SBTI) or FSLLRY, a protease-activated receptor-2 (PAR2) antagonist.Key findingsAcute stress significantly increased CPP, proteolytic and trypsin activities, and MMCP-1 levels. Atropine inhibited stress-induced impairment of CPP and strongly diminished total proteolytic and trypsin activities in stressed animals, but not MMCP-1 levels. Colonic contents from stressed animals increased CPP in mice tissues, this effect being inhibited by SBTI and PAR2 antagonist.SignificanceAcute stress activates cholinergic pathways, to trigger exocrine pancreatic secretion. Trypsin, released in these conditions, may be responsible for colonic barrier alterations through the activation of PAR2.     

Chronic peripheral administration of corticotropin-releasing factor causes colonic barrier dysfunction similar to psychological stress

Chronic psychological stress causes intestinal barrier dysfunction and impairs host defense mechanisms mediated by corticotrophin-releasing factor (CRF) and mast cells; however, the exact pathways involved are unclear. Here we investigated the effect of chronic CRF administration on colonic permeability and ion transport functions in rats and the role of mast cells in maintaining the abnormalities. CRF was delivered over 12 days via osmotic minipumps implanted subcutaneously in wild-type (+/+) and mast cell-deficient (Ws/Ws) rats. Colonic segments were excised for ex vivo functional studies in Ussing chambers [short-circuit current (Isc), conductance (G), and macromolecular permeability (horseradish peroxidase flux)], and analysis of morphological changes (mast cell numbers and bacterial host-interactions) was determined by light and electron microscopy. Chronic CRF treatment resulted in colonic mucosal dysfunction with increased Isc, G, and horseradish peroxidase flux in+/+but not in Ws/Ws rats. Furthermore, CRF administration caused mast cell hyperplasia and abnormal bacterial attachment and/or penetration into the mucosa only in+/+rats. Finally, selective CRF agonist/antagonist studies revealed that stimulation of CRF-R1 and CRF-R2 receptors induced the elevated secretory state and permeability dysfunction, respectively. Chronic CRF causes colonic barrier dysfunction in rats, which is mediated, at least in part, via mast cells. This information may be useful in designing novel treatment strategies for stress-related gastrointestinal disorders.     

Peripheral CRF activates myenteric neurons in the proximal colon through CRF(1) receptor in conscious rats

Corticotropin-releasing factor (CRF) injected peripherally induces clustered spike-burst activity in the proximal colon through CRF(1) receptors in rats. We investigated the effect of intraperitoneal CRF on proximal colon ganglionic myenteric cell activity in conscious rats using Fos immunohistochemistry on the colonic longitudinal muscle/myenteric plexus whole mount preparation. In vehicle-pretreated rats, there were only a few Fos immunoreactive (IR) cells per ganglion (1.2 +/- 0.6). CRF (10 microg/kg ip) induced Fos expression in 19.6 +/- 2.1 cells/ganglion. The CRF(1)/CRF(2) antagonist astressin (33 microg/kg ip) and the selective CRF(1) antagonist CP-154,526 (20 mg/kg sc) prevented intraperitoneal CRF-induced Fos expression in the proximal colon (number of Fos-IR cells/ganglion: 2.7 +/- 1.2 and 1.0 +/- 1.0, respectively), whereas atropine (1 mg/kg sc) had no effect. Double labeling of Fos with protein gene product 9.5 revealed the neuronal identity of activated cells that were encircled by varicose fibers immunoreactive to vesicular acetylcholine transporter. Fos immunoreactivity was mainly present in choline acetyltransferase-IR nerve cell bodies but not in the NADPH-diaphorase-positive cells. These results indicate that peripheral CRF activates myenteric cholinergic neurons in the proximal colon through CRF(1) receptor.     

Peripherally administered CRF stimulates colonic motility via central CRF receptors and vagal pathways in conscious rats

Corticotropin releasing factor (CRF) is one of the most important factors in the mechanism of stress-induced stimulation of colonic motility. However, it is controversial whether stress-induced stimulation of colonic motility is mediated via central or peripheral CRF receptors. We investigated the hypothesis that peripherally injected CRF accelerates colonic motility through the central CRF receptor, but not the peripheral CRF receptor. A strain gauge transducer was sutured on the serosal surface of the proximal colon. Colonic motility was monitored before and after the peripheral injection of CRF. An in vitro muscle strip study was also performed to investigate the peripheral effects of CRF. Subcutaneous injection of CRF (30-100 microg/kg) stimulated colonic motility in a dose-dependent manner. The stimulatory effect of peripherally administered CRF on colonic motility was abolished by truncal vagotomy, hexamethonium, atropine, and intracisternal injection of astressin (a CRF receptor antagonist). No responses to CRF (10(-9) -10(-7) M) of the muscle strips of the proximal colon were observed. These results suggest that the stimulatory effect of colonic motility in response to peripheral administration of CRF is mediated by the vagus nerve, nicotinic receptors, muscarinic receptors, and CRF receptors of the brain stem. It is concluded that peripherally administered CRF reaches the area postrema and activates the dorsal nucleus of vagi via central CRF receptors, resulting in stimulation of the vagal efferent and cholinergic transmission of the proximal colon.     

Stress signaling pathways activated by weaning mediate intestinal dysfunction in the pig

Weaning in the piglet is a stressful event associated with gastrointestinal disorders and increased disease susceptibility. Although stress is thought to play a role in postweaning intestinal disease, the mechanisms by which stress influences intestinal pathophysiology in the weaned pig are not understood. The objectives of these experiments were to investigate the impact of weaning on gastrointestinal health in the pig and to assess the role of stress signaling pathways in this response. Nineteen-day-old pigs were weaned, and mucosal barrier function and ion transport were assessed in jejunal and colonic tissues mounted on Ussing chambers. Weaning caused marked disturbances in intestinal barrier function, as demonstrated by significant (P < 0.01) reductions in transepithelial electrical resistance and increases in intestinal permeability to [3H]mannitol in both the jejunum and colon compared with intestinal tissues from age-matched, unweaned control pigs. Weaned intestinal tissues exhibited increased intestinal secretory activity, as demonstrated by elevated short-circuit current that was sensitive to treatment with tetrodotoxin and indomethacin, suggesting activation of enteric neural and prostaglandin synthesis pathways in weaned intestinal tissues. Western blot analyses of mucosal homogenates showed increased expression of corticotrophin-releasing factor (CRF) receptor 1 in the jejunum and colon of weaned intestinal tissues. Pretreatment of pigs with the CRF receptor antagonist alpha-helical CRF(9-41), which was injected intraperitoneally 30 min prior to weaning, abolished the stress-induced mucosal changes. Our results indicate that weaning stress induces mucosal dysfunction mediated by intestinal CRF receptors and activated by enteric nerves and prostanoid pathways.     

Corticotropin-releasing factor receptor 1 mediates acute and delayed stress-induced visceral hyperalgesia in maternally separated Long-Evans rats

In rodents, maternal pup interactions play an important role in programming the stress responsiveness of the adult organism. The aims of this study were 1) to determine the effect of different neonatal rearing conditions on acute and delayed stress-induced visceral sensitivity as well as on other measures of stress sensitivity of the adult animal; and 2) to determine the role of corticotropin-releasing factor receptor (CRF-R) subtype 1 (CRF(1)R) in mediating visceral hypersensitivity. Three groups of male Long-Evans rat pups were used: separation from their dam for 180 min daily from postnatal days 2-14 (MS180), daily separation (handling) for 15 min (H), or no handling. The visceromotor responses (VMR) to colorectal distension, stress-induced colonic motility, and anxiety-like behavior were assessed in the adult rats. The VMR was assessed at baseline, immediately after a 1-h water avoidance (WA) stress, and 24 h poststress. Astressin B, a nonselective CRF-R antagonist, or CP-154,526, a selective CRF(1)R antagonist, was administered before the stressor and/or before the 24-h measurement. MS rats developed acute and delayed stress-induced visceral hyperalgesia. In contrast, H rats showed hypoalgesia immediately after WA and no change in VMR on day 2. MS rats with visceral hyperalgesia also exhibited enhanced stress-induced colonic motility and increased anxiety-like behavior. In MS rats, both CRF-R antagonists abolished acute and delayed increases in VMR. Rearing conditions have a significant effect on adult stress responsiveness including immediate and delayed visceral pain responses to an acute stressor. Both acute and delayed stress-induced visceral hypersensitivity in MS rats are mediated by the CRF/CRF(1)R system.     

Nonopioid Effect of Morphine on Electrically Evoked Acetylcholine Release from Torpedo Electromotor Neurons

The release of acetylcholine from Torpedo electric organ slices following their electrical stimulation was modulated by morphine, by the muscarinic antagonist atropine, and by the nicotinic antagonist tubocurarine. Addition of either atropine or tubocurarine in the presence of the acetylcholinesterase inhibitor phospholine iodide enhanced acetylcholine release. The effects of the two antagonists were additive, a result suggesting that the secreted acetylcholine regulates its own release by activating both muscarinic and nicotinic cholinergic receptors and that these receptors inhibit acetylcholine release by different mechanisms. The effects of opiates on acetylcholine release were examined under conditions in which the cholinergic modulation of release is blocked, i.e., in the presence of atropine and tubocurarine. These experiments revealed that electrically evoked release of acetylcholine is blocked by the opiate agonists morphine and levorphanol. However, the inhibitory effect of morphine on acetylcholine release was not reversed by the opioid antagonist naloxone. Furthermore, dextrorphan, the nonopioid stereoisomer of levorphanol, had the same inhibitory effect as its opioid counterpart. These findings suggest that the effects of opiates on electrically evoked release of acetylcholine are not mediated by opioid receptors. The possible mechanisms underlying these nonopioid effects of morphine and levorphanol are discussed.     

Stress-Derived Corticotropin Releasing Factor Breaches Epithelial Endotoxin Tolerance

Background and aims

Loss of the endotoxin tolerance of intestinal epithelium contributes to a number of intestinal diseases. The etiology is not clear. Psychological stress is proposed to compromise the intestinal barrier function. The present study aims to elucidate the role of the stress-derived corticotropin releasing factor (CRF) in breaching the established intestinal epithelial endotoxin tolerance.

Methods

Epithelial cells of HT-29, T84 and MDCK were exposed to lipopolysaccharide to induce the endotoxin tolerance; the cells were then stimulated with CRF. The epithelial barrier function was determined using as indicators of the endotoxin tolerant status. A water-avoid stress mouse model was employed to test the role of CRF in breaching the established endotoxin tolerance in the intestine.

Results

The established endotoxin tolerance in the epithelial cell monolayers was broken down by a sequent exposure to CRF and LPS manifesting a marked drop of the transepithelial resistance (TER) and an increase in the permeability to a macromolecular tracer, horseradish peroxidase (HRP). The exposure to CRF also increased the expression of Cldn2 in the epithelial cells, which could be mimicked by over expression of TLR4 in epithelial cells. Over expression of Cldn2 resulted in low TER in epithelial monolayers and high permeability to HRP. After treating mice with the 10-day chronic stress, the intestinal epithelial barrier function was markedly compromised, which could be prevented by blocking either CRF, or TLR4, or Cldn2.

Conclusions

Psychological stress-derived CRF can breach the established endotoxin tolerance in the intestinal mucosa.     

Muscarinic receptors involved in airway vascular leakage induced by experimental gastro-oesophageal reflux

Gastro-oesophageal acid reflux may cause airway responses such as cough, bronchoconstriction and inflammation in asthmatic patients. Studies in humans or in animals have suggested that these responses involve cholinergic nerves. The purpose of this study was to investigate the role of the efferent vagal component on airway microvascular leakage induced by instillation of hydrochloric acid (HCl) into the oesophagus of guinea-pigs and the subtype of muscarinic receptors involved. Airway microvascular leakage induced by intra-oesophageal HCl instillation was abolished by bilateral vagotomy or by the nicotinic receptor antagonist, hexamethonium. HCl-induced leakage was inhibited by pretreatment with atropine, a non-specific muscarinic receptor antagonist, and also by pretreatment with either pirenzepine, a muscarinic M(1) receptor antagonist, or 4-DAMP, a muscarinic M(3) receptor antagonist. Pirenzepine was more potent than atropine and 4-DAMP. These antagonists were also studied on airway microvascular leakage or bronchoconstriction induced by intravenous administration of acetylcholine (ACh). Atropine, pirenzepine and 4-DAMP inhibited ACh-induced airway microvascular leakage with similar potencies. In sharp contrast, 4-DAMP and atropine were more potent inhibitors of ACh-induced bronchoconstriction than pirenzepine. Methoctramine, a muscarinic M(2) receptor antagonist, was ineffective in all experimental conditions. These results suggest that airway microvascular leakage caused by HCl intra-oesophageal instillation involves ACh release from vagus nerve terminals and that M(1) and M(3) receptors play a major role in cholinergic-mediated microvascular leakage, whereas M(3) receptors are mainly involved in ACh-induced bronchoconstriction.     

Role for IL-4 in macromolecular transport across human intestinal epithelium

Increased epithelial permeability is associated with intestinalinflammation, but there is little information on factors that regulatebarrier function in the absence of or before inflammation. We examinedif interleukin (IL)-4, or serum from atopic individuals, could alterthe barrier function of human colonic epithelial (T84) monolayers toantigenic-sized macromolecules. IL-4 and atopic serum significantlydecreased T84 monolayer resistance and increased transepithelialhorseradish peroxidase (HRP) transport. Bidirectional transport studiesdemonstrated that IL-4 selectively enhanced apical-to-basal movement ofHRP. HRP transport induced by IL-4 was inhibited by cold (4°C) andthe tyrosine kinase inhibitor genistein, but not the protein kinase Cinhibitor staurosporine. Electron microscopic analysis demonstratedthat both transcellular and paracellular pathways were affected.Anti-IL-4 antibodies abolished the increase in HRP transport inresponse to both IL-4 and serum. We speculate that enhanced productionof IL-4 in allergic conditions may be a predisposing factor toinflammation by allowing uptake of luminal antigens that gain access tothe mucosal immune system.     

Increased colonic sodium absorption in rats with chronic renal failure is partially mediated by AT1 receptor agonism

To test the hypothesis that colonic Na(+) transport is altered in the 5/6 nephrectomized rat model of chronic renal failure (CRF), we measured Na(+) fluxes across distal colon from control (CON), CRF, and CRF rats treated with the angiotensin II (ANG II) receptor antagonist losartan (+LOS). We also evaluated overall fluid and Na(+) balance and compared colonic protein and mRNA expression profiles for electroneutral [sodium-hydrogen exchanger (NHE)] and electrogenic Na(+) transport [epithelial sodium channel (ENaC)] in these groups. Consistent with a 60% enhancement in colonic Na(+) absorption in CRF, urinary Na(+) excretion increased by about 50% while serum Na(+) homeostasis was maintained. These CRF-induced changes in Na(+) handling were normalized by treatment with LOS. Net Na(+) absorption was also stimulated in in vitro tissues from CON rats following acute serosal addition of ANG II (10(-7) M), and this increase was blocked by AT(1) antagonism but not by an AT(2) antagonist. In CRF, colonic protein and mRNA expression variably increased for apical NHE2, NHE3, and ENaC alpha-, beta-, gamma-subunits, whereas expression of basolateral NHE1 and Na(+)-K(+)-ATPase (alpha-isoform) remained unaltered. Upregulation of the ENaC subunit mRNA was attenuated somewhat by LOS treatment. Previously, we showed that colonic AT(1) receptor protein is upregulated twofold in CRF, and here we find that AT(1) and AT(2) mRNA and AT(2) protein abundance is unchanged in CRF. We conclude that Na(+) absorption in CRF rat distal colon is increased due to elevated expression of proteins mediating electroneutral and electrogenic uptake and that it is partially mediated by AT(1) receptors.     

Antilaminin IgG releases TXB2 through activation of the cholinergic system

Antilaminin IgG was bound to cholinergic muscarinic receptors of normal mice heart and released TXB2, simulating the biological effect of a cholinergic agonist. Antilaminin IgG interfered with the binding of the radiolabelled muscarinis antagonist (-)3H-QNB in a noncompetitive fashion. Following the interaction of the antibody with the cholinergic receptor, an increased production of TXB2 occurred. This effect required the activation of the muscarinic cholinergic system, because it was blunted by atropine and mimicked by acetylcholine.     

Enhanced pelvic responses to stressors in female CRF-overexpressing mice

Acute stress affects gut functions through the activation of corticotropin-releasing factor (CRF) receptors. The impact of acute stress on pelvic viscera in the context of chronic stress is not well characterized. We investigated the colonic, urinary, and locomotor responses monitored as fecal pellet output (FPO), urine voiding, and ambulatory activity, respectively, in female and male CRF-overexpressing (CRF-OE) mice, a chronic stress model, and their wild-type littermates (WTL). Female CRF-OE mice, compared with WTL, had enhanced FPO to 2-min handling (150%) and 60-min novel environment (155%) but displayed a similar response to a 60-min partial restraint stress. Female CRF-OE mice, compared with WTL, also had a significantly increased number of urine spots (7.3 +/- 1.4 vs. 1.3 +/- 0.8 spots/h) and lower locomotor activity (246.8 +/- 47.8 vs. 388.2 +/- 31.9 entries/h) to a novel environment. Male CRF-OE mice and WTL both responded to a novel environment but failed to show differences between them in colonic and locomotor responses. Male WTL, compared with female WTL, had higher FPO (113%). In female CRF-OE mice, the CRF(1)/CRF(2) receptor antagonist astressin B and the selective CRF(2) receptor agonist mouse urocortin 2 (injected peripherally) prevented the enhanced defecation without affecting urine or locomotor responses to novel environment. RT-PCR showed that CRF(1) and CRF(2) receptors are expressed in the mouse colonic tissues. The data show that chronic stress, due to continuous central CRF overdrive, renders female CRF-OE mice to have enhanced pelvic and altered behavioral responses to superimposed mild stressors and that CRF(1)-initiated colonic response is counteracted by selective activation of CRF(2) receptor.     

Modulation of cerebral microvascular permeability by endothelial nicotinic acetylcholine receptors

Nicotine increases the permeability of the blood-brain barrier in vivo. This implies a possible role for nicotinic acetylcholine receptors in the regulation of cerebral microvascular permeability. Expression of nicotinic acetylcholine receptor subunits in cerebral microvessels was investigated with immunofluorescence microscopy. Positive immunoreactivity was found for receptor subunits alpha3, alpha5, alpha7, and beta2, but not subunits alpha4, beta3, or beta4. Blood-brain barrier permeability was assessed via in situ brain perfusion with [14C]sucrose. Nicotine increased the rate of sucrose entry into the brain from 0.3 +/- 0.1 to 1.1 +/- 0.2 microl.g(-1).min(-1), as previously described. This nicotine-induced increase in blood-brain barrier permeability was significantly attenuated by both the blood-brain barrier-permeant nicotinic antagonist mecamylamine and the blood-brain barrier-impermeant nicotinic antagonist hexamethonium to 0.5 +/- 0.2 and 0.3 +/- 0.2 microl.g(-1).min(-1), respectively. These data suggest that nicotinic acetylcholine receptors expressed on the cerebral microvascular endothelium mediate nicotine-induced changes in blood-brain barrier permeability.     

Mechanism of inhibitory action of peptide YY on cholecystokinin-induced contractions of isolated dog ileum

In isolated canine ileal longitudinal muscle preparations, cholecystokinin-octapeptide (CCK-8) produced a concentration-dependent contraction, which was suppressed by peptide YY (PYY) and was abolished by tetrodotoxin and atropine. PYY was approximately 2200-times as potent as CR1505, a CCK-receptor antagonist. PYY opposed the action of CCK-8 to a greater extent than that of nicotine and transmural electrical stimulation. Acetylcholine-induced contractions were not influenced by PYY. It seems likely that the CCK-8-induced ileal muscle contraction is associated with an activation of CCK receptors in cholinergic nerves, which generates nerve action potentials and releases acetylcholine, whereas CCK-8 acts on CCK receptors in gallbladder smooth muscle, producing contractions. It may be concluded that PYY inhibits the action of CCK-8 on ileal muscle strips, by inhibiting the release of acetylcholine from cholinergic nerve terminals. On the other hand, in the gallbladder, PYY does not appear to block cholinergic nerve function.     

Saturable (3H)cocaine binding in central nervous system of mouse

(³H)Cocaine was bound saturably to mouse brain membrane preparations, with a dissociation constant (K_d) of 0.6 μM and a maximal binding capacity of 3 pmol/mg of membrane protein. Binding was virtually maximal at 2°C, was sodium-insensitive, and was distributed rather uniformly throughout the brain. No, or only slight, displacing activities were observed for the neuro-transmitters norepinephrine, dopamine, acetylcholine, serotonin, and GABA, and for nicotine (nicotinic cholinergic receptor agonist), tubocurarine (nicotinic cholinergic receptor antagonist), morphine (opiate receptor agonist), and naloxone (opiate receptor antagonist). The cocaine analogs WIN 35,065-2 and WIN 35,428, which have enhanced stimulatory potency as compared with cocaine and only 15% of its local anesthetic activity, had affinities for the binding site similar to the affinity of cocaine itself. Displacing activities between 1 and 2 orders of magnitude weaker than those of cocaine itself were displayed by the local anesthetics, d-amphetamine, decamethonium, and atropine.     

Octreotide-induced drinking, vasopressin, and pressure responses: role of central angiotensin and ACh

The involvement of central angiotensinergic and cholinergic mechanisms in the effects of the intracerebroventricularly injected somatostatin analog octreotide (Oct) on drinking, blood pressure, and vasopressin secretion in the rat was investigated. Intracerebroventricular Oct elicited prompt drinking lasting for 10 min. Water consumption depended on the dose of Oct (0.01, 0.1, and 0. 4 microgram). The drinking response to Oct was inhibited by pretreatments with the intracerebroventricularly injected angiotensin-converting enzyme inhibitor captopril, the AT(1)/AT(2) angiotensin receptor antagonist saralasin, the selective AT(1) receptor antagonist losartan, or the muscarinic cholinergic receptor antagonist atropine. The dipsogenic effect of Oct was not altered by prior subcutaneous injection of naloxone. Oct stimulated vasopressin secretion and enhanced blood pressure. These responses were also blocked by pretreatments with captopril or atropine. Previous reports indicate that the central angiotensinergic and cholinergic mechanisms stimulate drinking and vasopressin secretion independently. We suggest that somatostatin acting on sst2 or sst5 receptors modulates central angiotensinergic and cholinergic mechanisms involved in the regulation of fluid balance.     

CRF R1受体拮抗剂对幼年大鼠快眼动睡眠的影响

目的：观察腹腔注射CRFR1受体拮抗剂NBI27914对幼年大鼠睡眠/觉醒周期的影响。方法：生后13d的大鼠行电极安装术,术后12h开始多导睡眠描记,描记的第6h分别给予腹腔注射不同剂量的NBI27914、阿托品和等量生理盐水。结果：与基础水平相比,腹腔注射任何剂量NBI27914使幼年大鼠快眼动（REM）睡眠均显著减少,同时伴随着非快眼动（NREM）睡眠的增加,而生理盐水组无显著变化;阿托品组的大鼠REM睡眠也显著减少,伴随的是觉醒的增加。结论：阻断CRFR1受体可以剥夺幼鼠的REM睡眠。     

Effects of acetylcholine on sodium-dependent high-affinity glutamate transport in rat striatal homogenates

Incubation of rat striatal tissue in the presence of acetylcholine, carbachol, oxotremorine, or nicotine results in a significant decrease in the sodium-dependent high-affinity glutamate uptake (HAGU). The cholinergic inhibitory effect on glutamate transport is no more detectable in the presence of atropine, a cholinergic receptor antagonist. These data support the hypothesis that glutamatergic nerve ending activity in the striatum is modulated by cholinergic neurons. The effects would involve both muscarinic and nicotinic presynaptic receptors located on the corticostriatal glutamatergic terminals.