Effect of CCK pretreatment on the CCK sensitivity of rat polymodal gastric vagal afferent in vitro

To prevent the blood-borne interference and reflex actions via neighboring organs and the central nervous system, the study was conducted in an in vitro isolated stomach-gastric vagus nerve preparation obtained from overnight-fasted, urethan-anesthetized rats. Afferent unit action potentials were recorded from the gastric branch of the vagus nerve. The left gastric artery was catheterized for intra-arterial injection. In vitro we found that 1) 55/70 gastric vagal afferents (GVAs) were polymodal, responding to CCK-8 and mechanical stimuli, 13 were mechanoreceptive, and 2 were CCK-responsive; 2) sequential or randomized intra-arterial injections of CCK-8 (0.1-200 pmol) dose-dependently increased firing rate and reached the peak rate at 100 pmol; 3) the action was suppressed by CCK-A (Devazepide) but not by CCK-B (L-365,260) receptor antagonist; 4) neither antagonist blocked the mechanosensitivity of GVA fibers. These results are consistent with corresponding in vivo well-documented findings. Histological data indicate that the layered structure of the stomach wall was preserved in vitro for 6-8 h. Based on these results, it seems reasonable to use the in vitro preparation for conducting a study that is usually difficult to be performed in vivo. For instance, because there was no blood supply in vitro, the composition of the interstitial fluid, i.e., the ambient nerve terminals, can be better controlled and influenced by intra-arterial injection of a defined solution. Here we report that acutely changing the ambient CCK level by a conditioning stimulus (a preceding intra-arterial injection of increasing doses of CCK-8) reduced the CCK sensitivity of GVA terminals to a subsequent test stimulus (a constant dose of CCK-8 intra-arterial injection).     

Mechanisms of action of CCK to activate central vagal afferent terminals

Cholecystokinin [CCK] is a peptide released as a hormone by the proximal gut in response to the presence of peptones and fatty acid in the gut. Considerable evidence suggests that CCK inhibits feeding behavior and gastric function by acting as a paracrine modulator of vagal afferents in the periphery, especially in the duodenum. CCK is also widely distributed throughout the mammalian brain and appears to function as a neurotransmitter and neuromodulator. More recent studies have suggested that CCK may act directly within the CNS to activate central vagal afferent terminal inputs to the solitary nucleus. We have developed an in vitro calcium imaging method that reveals, for the first time, the direct effects of this peptide on vagal terminals in the solitary nucleus. In vitro imaging reveals that CCK provokes increases in intracellular calcium in vagal afferent terminals as a consequence of a complex interaction between protein kinase A [PKA] and phospholipase C [PLC] transduction mechanisms that open L-type calcium channels and causes endoplasmic reticular [ER] calcium release. The subsequent activation of PKC may be responsible for initiating calcium spiking which is dependent on a TTX-sensitive mechanism. Thus, imaging of the isolated but spatially intact hindbrain slice has allowed a more complete appreciation of the interdependent transduction mechanisms used by CCK to excite identified central vagal afferent fibers and varicosities.     

Ileal short-chain fatty acids inhibit gastric motility by a humoral pathway

The aim of this study was to evaluate the nervous and humoral pathways involved in short-chain fatty acid (SCFA)-induced ileal brake in conscious pigs. The role of extrinsic ileal innervation was evaluated after SCFA infusion in innervated and denervated Babkin's ileal loops, and gastric motility was measured with strain gauges. Peptide YY (PYY) and glucagon-like peptide-1 (GLP-1) concentrations were evaluated in both situations. The possible involvement of absorbed SCFA was tested by using intravenous infusion of acetate. Ileal SCFA infusion in the intact terminal ileum decreased the amplitude of distal and terminal antral contractions (33 +/- 1.2 vs. 49 +/- 1.2% of the maximal amplitude recorded before infusion) and increased their frequency (1.5 +/- 0.11 vs. 1.3 +/- 0.10/min). Similar effects were observed during SCFA infusion in ileal innervated and denervated loops (amplitude, 35 +/- 1.0 and 34 +/- 0. 8 vs. 47 +/- 1.3 and 43 +/- 1.2%; frequency, 1.4 +/- 0.07 and 1.6 +/- 0.06 vs. 1.1 +/- 0.14 and 1.0 +/- 0.12/min). Intravenous acetate did not modify the amplitude and frequency of antral contractions. PYY but not GLP-1 concentrations were increased during SCFA infusion in innervated and denervated loops. In conclusion, ileal SCFA inhibit distal gastric motility by a humoral pathway involving the release of an inhibiting factor, which is likely PYY.     

Leptin inhibits gastric emptying in rats: role of CCK receptors and vagal afferent fibers

Leptin regulates energy homeostasis and body weight by balancing energy intake and expenditure. It was recently reported that leptin, released into the gut lumen during the cephalic phase of gastric secretion, is capable of initiating intestinal nutrient absorption. Vagal afferent neurons also express receptors for both CCK and leptin, which are believed to interact in controlling food intake. The present study was undertaken to investigate the central and peripheral effects of leptin on gastric emptying rate. Under anesthesia, male Sprague-Dawley rats (250-300 g) were fitted with gastric Gregory cannulas (n=12) and some had additional cerebroventricular cannulas inserted into their right lateral ventricles. Following recovery, the rate of gastric emptying of saline (300 mOsm/kg H(2)O) was determined after instillation into the gastric fistula (3 ml, 37 degrees C, containing phenol red, 60 mg/l as a non-absorbable dilution marker). Gastric emptying rate was determined from the volume and phenol red concentrations recovered after 5 min. Leptin, injected intraperitoneally (i.p.; 10, 30, 60, 100 microg/kg) or intracerebroventricularly (i.c.v.; 5, 15 microg/rat) 15 min before the emptying, delayed gastric emptying rate of saline at the dose of 30 microg/kg or 15 microg/rat (p<0.001). When CCK(1) receptor blocker L-364,718 (1 mg/kg, i.p.), CCK(2) receptor blocker L-365,260 (1 mg/kg, ip) or adrenergic ganglion blocker bretylium tosylate (15 mg/kg, i.p.) was administered 15 min before ip leptin (30 microg/kg) injections, leptin-induced delay in gastric emptying was abolished only by the CCK(1) receptor blocker (p<0.001). However, the inhibitory effect of central leptin on gastric emptying was reversed by adrenergic blockade, but not by either CCK antagonists. Our results demonstrated that leptin delays gastric emptying. The peripheral effect of leptin on gastric motility appears to be mediated by CCK(1) receptors, suggesting the release of CCK and the involvement of vagal afferent fibers. On the other hand, the central effect of leptin on gastric emptying is likely to be mediated by adrenergic neurons. These results indicate the existence of a functional interaction between leptin and CCK receptors leading to inhibition of gastric emptying and short-term suppression of food intake, providing an additional feedback control in producing satiety.     

CCK enhances response to gastric distension by acting on capsaicin-insensitive vagal afferents

Capsaicin treatment destroys vagal afferent C fibers and markedly attenuates reduction of food intake and induction of hindbrain Fos expression by CCK. However, both anatomical and electrophysiological data indicate that some gastric vagal afferents are not destroyed by capsaicin. Because CCK enhances behavioral and electrophysiological responses to gastric distension in rats and people, we hypothesized that CCK might enhance the vagal afferent response to gastric distension via an action on capsaicin-insensitive vagal afferents. To test this hypothesis, we quantified expression of Fos-like immunoreactivity (Fos) in the dorsal vagal complex (DVC) of capsaicin-treated (Cap) and control rats (Veh), following gastric balloon distension alone and in combination with CCK injection. In Veh rats, intraperitoneal CCK significantly increased DVC Fos, especially in nucleus of the solitary tract (NTS), whereas in Cap rats, CCK did not significantly increase DVC Fos. In contrast to CCK, gastric distension did significantly increase Fos expression in the NTS of both Veh and Cap rats, although distension-induced Fos was attenuated in Cap rats. When CCK was administered during gastric distension, it significantly enhanced NTS Fos expression in response to distension in Cap rats. Furthermore, CCK's enhancement of distension-induced Fos in Cap rats was reversed by the selective CCK-A receptor antagonist lorglumide. We conclude that CCK directly activates capsaicin-sensitive C-type vagal afferents. However, in capsaicin-resistant A-type afferents, CCK's principal action may be facilitation of responses to gastric distension.     

Apolipoprotein A-IV, a putative satiety/antiatherogenic factor, rises after gastric bypass

Roux-en-Y gastric bypass surgery (RYGBP) leads to improvements in satiety and obesity-related comorbidities. The mechanism(s) underlying these improvements are not known but may be revealed in part by discovery proteomics. Therefore, fasting plasma was collected from 12 subjects (mean BMI >45) during RYGBP and during a second procedure approximately 17 months later. Body weight, obesity-related comorbidities, and medication use were decreased after RYGBP. Mass spectrometry-based proteomic analysis was performed on a subset of seven samples using isobaric isotope-coded affinity tags (four plex iTRAQ). Initial proteomic analysis (n = 7) quantified and identified hundreds of plasma proteins. Manual inspection of the data revealed a 2.6 +/- 0.5-fold increase in apolipoprotein A-IV (apo A-IV, gene designation: APOA4), a approximately 46-kDa glycoprotein synthesized mainly in the bypassed small bowel and liver after RYGBP. The change in apo A-IV was significantly greater than other apolipoproteins. Immunoblot analysis of the full longitudinal sample set (n = 12) indicated even higher increases (8.3 +/- 0.2 fold) in apo A-IV. Thus iTRAQ may underestimate the changes in protein concentrations compared to western blotting of apo A-IV. Apo A-IV inhibits gastric emptying and serves as a satiety factor whose synthesis and secretion are increased by the ingestion of dietary fat. It also possesses anti-inflammatory and antiatherogenic properties. Based on these functions, we speculate changes in apo A-IV may contribute to weight loss as well as the improvements in inflammation and cardiovascular disease after RYGBP. In addition, the findings provide evidence validating the use of iTRAQ proteomics in discovery-based studies of post-RYGBP improvements in obesity-related medical comorbidities.     

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.     

Nesfatin-1 inhibits gastric acid secretion via a central vagal mechanism in rats

Nesfatin-1, a novel hypothalamic peptide, inhibits nocturnal feeding behavior and gastrointestinal motility in rodents. The effects of nesfatin-1 on gastrointestinal secretory function, including gastric acid production, have not been evaluated. Nesfatin-1 was injected into the fourth intracerebral ventricle (4V) of chronically cannulated rats to identify a nesfatin dose sufficient to inhibit food intake. Nesfatin-1 (2 μg) inhibited dark-phase food intake, in a dose-dependent fashion, for >3 h. Gastric acid production was evaluated in urethane-anesthetized rats. Nesfatin-1 (2 μg) was introduced via the 4V following endocrine stimulation of gastric acid secretion by pentagastrin (2 μg·kg(-1)·h(-1) iv), vagal stimulation with 2-deoxy-d-glucose (200 mg/kg sc), or no stimulus. Gastric secretions were collected via gastric cannula and neutralized by titration to determine acid content. Nesfatin-1 did not affect basal and pentagastrin-stimulated gastric acid secretion, whereas 2-deoxy-d-glucose-stimulated gastric acid production was inhibited by nesfatin-1 in a dose-dependent manner. c-Fos immunofluorescence in brain sections was used to evaluate in vivo neuronal activation by nesfatin-1 administered via the 4V. Nesfatin-1 caused activation of efferent vagal neurons, as evidenced by a 16-fold increase in the mean number of c-Fos-positive neurons in the dorsal motor nucleus of the vagus (DMNV) in nesfatin-1-treated animals vs. controls (P < 0.01). Finally, nesfatin-induced Ca(2+) signaling was evaluated in primary cultured DMNV neurons from neonatal rats. Nesfatin-1 caused dose-dependent Ca(2+) increments in 95% of cultured DMNV neurons. These studies demonstrate that central administration of nesfatin-1, at doses sufficient to inhibit food intake, results in inhibition of vagally stimulated secretion of gastric acid. Nesfatin-1 activates DMNV efferent vagal neurons in vivo and triggers Ca(2+) signaling in cultured DMNV neurons.     

Receptor subtype specific activation of the rat gastric vagal afferent fibers to serotonin

Systemic administration (i.v.) of serotonin (5-HT) evoked a transient vagal afferent nerve discharge, bradycardia, and hypotension in the rat. The half-effective dose of 5-HT for nerve discharge was 13 micro g/kg. The time- and dose-dependent kinetics of the nerve discharge rate were similar to the change of heart rate. The afferent neuronal discharge was mimicked by a selective 5-HT3 receptor agonist, 1-phenylbiguanide hydrochloride (PBA), and inhibited by a selective 5-HT3 antagonist, granisetron. The 5-HT(3/4) agonist, cisapride partially activated the vagus nerve, but the 5-HT4 agonist, RS6733 had no effect on the vagal afferent activity. Intra-gastric perfusion of lidocaine, moreover, abolished the 5-HT-induced vagal activation. These results indicate that the 5-HT transmission signal in the gastric mucosa inputs to the brain stem via 5-HT3 receptor-mediated vagal nerve afferent.     

迷走背核微量注射P物质抑制大鼠胃肌电活动和胃运动

本工作观察到大鼠迷走背核微量注射Ｐ物质（Ｓｕｂｓｔａｎｃｅ Ｐ,ＳＰ）抑制胃肌电快波和胃运动。该效应可分别迷走背核注射ＳＰ抗血清,ＳＰ受体拮抗剂「Ａｒｇ＾６,Ｄ－Ｔｒｐ＾７,９,Ｎ－Ｍｅ－Ｐｈｅ＾８」－ＳＰ６－１１或切断迷走神经所消除。用利血平耗竭交感神经递质则不影响该效应。     

Exogenous leptin inhibits the secretion of pancreatic juice via a duodenal CCK1-vagal-dependent mechanism in anaesthetized rats

Leptin originally described as product of the ob gene has been shown to be expressed in various tissues including the gastrointestinal tract. In this study, we investigated the influence of leptin on the secretion of pancreatic juice in biliary-pancreatic duct cannulated anaesthetised rats and in dispersed rat pancreatic acini in vitro. Exogenous leptin was given in boluses intravenously with or without CCK-8 (12 pmol kg(-1) body weight) in the presence or absence pharmacological CCK(1) receptor blockade, cervical vagotomy, and capsaicin pre-treatment. Administration of leptin (0.1, 1 and 10 microg kg(-1) body weight) did not affect the volume of bile and pancreatic juice while the protein and trypsin outputs were reduced in a dose-dependent manner. In the rats, leptin inhibited CCK-8 stimulated protein and trypsin outputs stronger than the basal pancreatic secretion. The inhibition by leptin was abolished by the pharmacological CCK(1) receptor blockade, cervical vagotomy, and capsaicin pre-treatment. In contrast, leptin did not affect basal and CCK-8-stimulated amylase release from the dispersed rat pancreatic acini in vitro. In conclusion, the results of the present study suggest that leptin does not act directly on the rat pancreatic acinar cells but inhibits the secretion of pancreatic enzymes acting indirectly via a neurohormonal CCK-vagal-dependent mechanism.     

Blue light stimulates cyanobacterial motility via a cAMP signal transduction system

The participation of cAMP in photosignal transduction in cyanobacteria was investigated. When cells of the cyanobacterium Synechocystis sp. PCC 6803 were exposed to light, cellular cAMP contents increased within a few minutes. Among incident monochromatic lights, blue light (450 nm) markedly increased cellular cAMP content, while red (630 nm) and far-red (720 nm) lights did not. Disruption of the cya1 gene encoding an adenylate cyclase caused the insensitivity of cellular cAMP level to blue light. Treatment of wild-type cells with the flavin antagonist phenylacetic acid inhibited this blue light effect. The motility of wild-type cells was enhanced by blue light, whereas that of cya1 mutant cells was not. Based on these results, we concluded that a blue light-cAMP signal transduction system stimulates the motility of Synechocystis sp. PCC 6803.     

Heat-stable enterotoxin of Escherichia coli stimulates a non-CFTR-mediated duodenal bicarbonate secretory pathway

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is an important pathway for duodenal mucosal bicarbonate secretion. Duodenal biopsies from CF patients secrete bicarbonate in response to heat-stable enterotoxin from Escherichia coli (STa) but not cAMP. To explore the mechanism of STa-induced bicarbonate secretion in CF more fully, we examined the role of CFTR in STa-stimulated duodenal bicarbonate secretion in mice. In vivo, the duodenum of CFTR (-/-) or control mice was perfused with forskolin (10(-4) M), STa (10(-7) M), uroguanylin (10(-7) M), 8-bromoguanosine 3',5'-cGMP (8-Br-cGMP) (10(-3) M), genistein (10(-6) M) plus STa, or herbimycin A (10(-6) M) plus STa. In vitro, duodenal mucosae were voltage-clamped in Ussing chambers, and bicarbonate secretion was measured by pH-stat. The effect of genistein, DIDS (10(-4) M), and chloride removal was also studied in vitro. Control, but not CF, mice produced a significant increase in duodenal bicarbonate secretion after perfusion with forskolin, uroguanylin, or 8-Br-cGMP. However, both control and CF animals responded to STa with significant increases in bicarbonate output. Genistein and herbimycin A abolished this response in CF mice but not in controls. In vitro, STa-stimulated bicarbonate secretion in CF tissues was inhibited by genistein, DIDS, and chloride-free conditions, whereas bicarbonate secretion persisted in control mice. In the CF duodenum, STa can stimulate bicarbonate secretion via tyrosine kinase activity resulting in apical Cl(-)/HCO(3)(-) exchange. Further studies elucidating the intracellular mechanisms responsible for such non-CFTR mediated bicarbonate secretion may lead to important therapies for CF.     

Caerulein, a cholecystokinin-related peptide, depresses somatic function via the vagal afferent system

Intravenous doses of 0.5-8 micrograms/kg (0.3-4.9 nmol/kg) of caerulein, a cholecystokinin-related peptide, depressed the crossed extensor reflex response of chloralose-anesthetized rats in a dose-dependent manner. Intraventricularly administered caerulein was effective only at high doses (1-2 micrograms/animal, i.e., 0.6-1.2 nmol/animal). Cervical vagotomy completely abolished the inhibitory effect of peripherally injected caerulein. Electrical stimulation of vagal afferent nerves could simulate the caerulein effect to some extent. These results suggest that the primary site of action of peripherally administered caerulein is located in the gastrointestinal tract and thus the generated afferent vagal impulses mediate the reflex depression via the nucleus tractus solitarius and other as yet unclarified brain stem structures.     

An afferent vagal nerve pathway links hepatic PPARalpha activation to glucocorticoid-induced insulin resistance and hypertension

Glucocorticoid excess causes insulin resistance and hypertension. Hepatic expression of PPARalpha (Ppara) is required for glucocorticoid-induced insulin resistance. Here we demonstrate that afferent fibers of the vagus nerve interface with hepatic Ppara expression to disrupt blood pressure and glucose homeostasis in response to glucocorticoids. Selective hepatic vagotomy decreased hyperglycemia, hyperinsulinemia, hepatic insulin resistance, Ppara expression, and phosphoenolpyruvate carboxykinase (PEPCK) enzyme activity in dexamethasone-treated Ppara(+/+) mice. Selective vagotomy also decreased blood pressure, adrenergic tone, renin activity, and urinary sodium retention in these mice. Hepatic reconstitution of Ppara in nondiabetic, normotensive dexamethasone-treated PPARalpha null mice increased glucose, insulin, hepatic PEPCK enzyme activity, blood pressure, and renin activity in sham-operated animals but not hepatic-vagotomized animals. Disruption of vagal afferent fibers by chemical or surgical means prevented glucocorticoid-induced metabolic derangements. We conclude that a dynamic interaction between hepatic Ppara expression and a vagal afferent pathway is essential for glucocorticoid induction of diabetes and hypertension.     

Sphingosine stimulates cellular proliferation via a protein kinase C-independent pathway

Sphingosine, a metabolite of membrane sphingolipids, is generally considered to be cytotoxic for a variety of cell types. However, we have found that sphingosine at low concentrations stimulates DNA synthesis and acts synergistically with known growth factors to induce proliferation of quiescent Swiss 3T3 fibroblasts. Structurally related analogs of sphingosine, such as N-stearoylsphingosine and other long chain aliphatic amines, had no mitogenic effects, suggesting that sphingosine did not induce nonspecific membrane perturbations. Sphingosine, which has been proposed to be a physiological inhibitor of protein kinase C, also markedly potentiates the mitogenic effect of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Sphingosine still stimulates DNA synthesis in cells made protein kinase C deficient by prolonged treatment with phorbol ester. At mitogenic concentrations, sphingosine does not bind to protein kinase C as shown by its lack of effect on phorbol dibutyrate binding. Only at higher concentrations, in the cytotoxic range, was there a displacement of phorbol dibutyrate from its cellular-binding sites. In contrast to sphingosine, H-7, a known inhibitor of protein kinase C, inhibited the mitogenic response to TPA and the TPA-induced phosphorylation of the 80 kDa cellular substrate of protein kinase C. Our results suggest that sphingosine may play an important role as a positive regulator of cell growth acting in a fundamentally different, protein kinase C-independent pathway.     

Oral administration of Tyr-MIF-1 stimulates gastric emptying and gastrointestinal motility in rodents.

The effects of orally administered Tyr-MIF-1, an agonist of an endogenous antiopiate system, were examined on gastric emptying in mice and gastrointestinal myoelectric activity in rats. Tyr-MIF-1 (5 mg/kg in mice, 20 mg/kg in rats) accelerated gastric emptying of a methylcellulose test meal, increased the frequency of antral spike bursts, and disrupted intestinal migrating myoelectric complexes. These effects were reproduced by a subcutaneous administration of Tyr-MIF-1 at the same dosage. They were blocked by naloxone (1 mg/kg) but not by the kappa receptor subtype antagonist MR 2266 (1 mg/kg). The GABAA antagonist bicuculline (0.5 mg/kg), but not the GABAB antagonist 2-hydroxysaclofen (4 mg/kg), also antagonized the effects of Tyr-MIF-1. These data demonstrate that oral Tyr-MIF-1 stimulates gastric emptying and gastrointestinal motility through a systemic or central action that involves opioid and GABA systems.