Increased susceptibility of rat gastric mucosa to ulcerogenic stimulation with aging

We examined the influences of aging on gastric damage and gastric mucosal blood flow (GMBF) responses induced by acid back-diffusion, following the barrier disruption, and investigated the relation of capsaicin-sensitive sensory nerves to these changes. Male Fischer rats 3, 13, and 24 months old were used. Under urethane anesthesia, a rat stomach was mounted on a chamber, and gastric potential difference (PD), luminal H⁺ loss, and GMBF were measured before, during, and after exposure to 20 mM sodium taurocholate (TC) for 30 min, in the presence of 50 mM HCl. Mucosal exposure to TC caused surface cell damage, PD reduction, and acid back-diffusion (luminal H⁺ loss) in all groups of rats; PD reduction and the amount of H⁺ loss were not significantly different between young and aged rats. In young rats, a marked increase of GMBF was observed with luminal acid loss following TC treatment, yet it resulted in less damage in the gastric mucosa. In aged rats, however, such GMBF responses were apparently mitigated, leading to a significant worsening of gastric mucosal lesions induced by TC. Mucosal application of capsaicin (0.1 mg/ml) caused an increase of GMBF in young rats, but this response was significantly attenuated in aged rats. In addition, the amount of calcitonin gene-related peptide (CGRP) released in the isolated stomach in response to capsaicin (1 × 10^–5 M) was significantly lower in aged animals when compared to young rats. These findings suggest that the gastric mucosa of aged rats is more vulnerable to acid back-diffusion following the barrier disruption, partly because of dysfunction of GMBF responses mediated by capsaicin-sensitive sensory neurons in the acidic conditions.     

Role of capsaicin-sensitive afferent neurons in mucosal blood flow response of rat stomach induced by mild irritants

The role of capsaicin-sensitive sensory nerves in gastric mucosal blood flow (GMBF) responses to mild irritants was investigated in the rat stomach mounted on a lucite chamber using hypertonic NaCl and 0.2 N HCl. Exposure of the mucosa to hypertonic NaCl (0.5, 0.75, 1 M) for 10 min caused a reduction in the transmucosal potential difference (PD) in a concentration-related manner, followed by an increase of luminal pH and GMBF. In contrast, mucosal application of 0.2 N HCl caused no or little change in PD and pH, but increased GMBF significantly. Functional ablation of capsaicin-sensitive sensory nerves significantly inhibited the increase of GMBF after exposure to these irritants, although the PD and pH responses induced by 1 M NaCl remained unaltered by this treatment. Pretreatment with indomethacin (5 mg/kg, subcutaneously) significantly attenuated the GMBF responses to 1 M NaCl and 0.2 N HCl and inhibited the increase of pH caused by 1 M NaCl. Mucosal application of capsaicin (0.1 mg/ml for 10 min) produced an increase of GMBF without being accompanied by change in PH and pH, and this effect was significantly blocked by either indomethacin or chemical deafferentation. These results suggest that capsaicin-sensitive sensory nerves as well as endogenous prostaglandins may be involved in the mechanism of GMBF responses induced by mild irritants, and the latter might sensitize these nerves to mucosal irritation. PD reduction may be obligatory for pH but not GMBF responses.     

Effect of lafutidine,a histamine H2-receptor antagonist,on gastric mucosal blood flow and duodenal HCO3- secretion in rats: relation to capsaicin-sensitive afferent neurons

Lafutidine is a new type of antiulcer drug, possessing both an antisecretory effect, exerted via a blockade of the histamine H₂ receptor, and gastroprotective activity, mediated by capsaicin-sensitive afferent nerves (CSN). In the present study, we examined the effect of lafutidine on gastric mucosal blood flow (GMBF) and duodenal HCO₃ ^– secretion (DAS) under basal and acid-stimulated conditions in rats. Under urethane anesthesia, GMBF was measured using a laser Doppler flowmeter in a chambered stomach before and after exposure to 20 mM taurocholate (TC) plus 50 mM HCl, while DAS was measured in a proximal duodenal loop before and after mucosal acidification (10 mM HCl for 10 min) by titrating the perfusate at pH 7.0 using a pH-stat method and by adding 10 mM HCl. Lafutidine given intraperitoneally affected neither GMBF nor DAS under basal conditions, but augmented an increase in both GMBF and DAS induced by mucosal acidification. Although the acid-induced GMBF and DAS responses were significantly mitigated by both indomethacin and sensory deafferentation but not by ruthenium red (RT), the vanilloid receptor (VR)-1 antagonist, the responses were preserved in lafutidine-treated animals, even in the presence of indomethacin. Both GMBF and DAS were significantly increased by local application of capsaicin, the responses being attenuated by indomethacin and RT as well as sensory deafferentation. Lafutidine augmented the GMBF and DAS responses to capsaicin and preserved the responses, even in the presence of indomethacin. Capsaicin evoked an increase in [Ca²⁺]_i in rat VR1-transfected HEK293 cells, while lafutidine had no effect by itself on [Ca²⁺]_i in these cells and did not affect the increase in [Ca²⁺]_i evoked by capsaicin. In conclusion, these results suggest that lafutidine mimics endogenous effects of prostaglandins to augment the GMBF and DAS responses to acid or capsaicin, probably by sensitizing CSN through an unknown site other than VR1. The luminal H⁺ itself is not a ligand for the RT-sensitive site of VR1 but plays a modulator role in the CSN-mediated physiological responses.     

Gastro-protective action of lafutidine mediated by capsaicin-sensitive afferent neurons without interaction with TRPV1 and involvement of endogenous prostaglandins

AIM: Lafutidine, a histamine H2 receptor antagonist, exhibits gastro-protective action mediated by capsaicin-sensitive afferent neurons (CSN). We compared the effect between lafutidine and capsaicin, with respect to the interaction with endogenous prostaglandins (PG), nitric oxide (NO) and the afferent neurons, including transient receptor potential vanilloid subtype 1 (TRPV1). METHODS: Male SD rats and C57BL/6 mice, both wild-type and prostacyclin IP receptor knockout animals, were used after 18 h of fasting. Gastric lesions were induced by the po administration of HCI/ethanol (60% in 150 mmol/L HCI) in a volume of 1 mL for rats or 0.3 mL for mice. RESULTS: Both lafutidine and capsaicin (1-10 mg/kg, po) afforded dose-dependent protection against HCI/ ethanol in rats and mice. The effects were attenuated by both the ablation of CSN and pretreatment with NG-nitro-L-arginine methyl ester, yet only the effect of capsaicin was mitigated by prior administration of capsazepine, the TRPV1 antagonist, as well as indomethacin. Lafutidine protected the stomach against HCI/ethanol in IP receptor knockout mice, similar to wild-type animals, while capsaicin failed to afford protection in the animals lacking IP receptors. Neither of these agents affected the mucosal PGE2 or 6-keto PGF1αcontents in rat stomachs. Capsaicin evoked an increase in [Ca2 ]i in rat TRPV1-transfected HEK293 cells while lafutidine did not. CONCLUSION: These results suggest that although both lafutidine and capsaicin exhibit gastro-protective action mediated by CSN, the mode of their effects differs regarding the dependency on endogenous PGs/IP receptors and TRPV1. It is assumed that lafutidine interacts with CSN at yet unidentified sites other than TRPV1.     

Mucosal irritative and healing impairment action of risedronate in rat stomachs: comparison with alendronate

BACKGROUND AND AIM: We used alendronate and risedronate as bisphosphonates and examined whether or not these agents have a mucosal irritative action in the stomach and impair the healing of pre-existing gastric ulcers in rats. METHODS: Male Sprague Dawley (SD) rats were used in the following two studies: (i) the effects of risedronate and alendronate on gastric potential difference (PD), gastric mucosal blood flow (GMBF) and acid back-diffusion in the stomach mounted on ex vivo chamber under urethane anesthesia and; (ii) the influence of daily treatment with these drugs on the healing of acetic acid-induced gastric ulcers was examined. RESULTS: Mucosal application of risedronate produced PD reduction in the saline-perfused stomachs in a dose-dependent manner. Alendronate also produced a marked PD reduction, the effect being more potent than that of risedronate. In the stomach exposed to acid (100 mM HCl), both drugs produced a marked reduction in PD, followed by acid back-diffusion and a small increase in GMBF, resulting in hemorrhagic lesions, and the effects again were more pronounced with alendronate. These irritative effects were dependent on the pH of drug solution and the action was more potent at pH 7 than pH 4. Conversely, the healing of acetic acid-induced gastric ulcers was significantly delayed by daily administration of these drugs, yet this effect was less pronounced in the case of risedronate. The healing impairing effect of these bisphosphonates was potentiated by coadministration of indomethacin. CONCLUSION: Both alendronate and risedronate have mucosal irritative and healing impairing effects in the stomach, yet the effect of risedronate was much less pronounced compared to alendronate. It is assumed that risedronate is safer than alendronate as the antiresorptive agent in patients with diseases related to bone remodeling.     

Prostaglandin E inhibits indomethacin-induced gastric lesions through EP-1 receptors

BACKGROUNDS AND AIMS: We examined the effect of various prostaglandin E (PGE) analogs specific to EP receptor subtypes on indomethacin-induced gastric lesions in rats and investigated which EP receptor subtype is involved in the protective action of PGE(2) using EP-receptor knockout mice. METHODS: Gastric lesions were induced by subcutaneous administration of indomethacin (35 mg/kg). Gastric motility was measured using a balloon method, while neutrophil chemotaxis determined using a Boyden chamber. RESULTS: Indomethacin-induced gastric lesions were significantly prevented by PGE(2) as well as atropine, and the former effect was mimicked by sulprostone (EP(1)/EP(3)) and 17-phenyl PGE(2) (EP(1)) and antagonized by an EP(1) antagonist, ONO-AE-829. Neither butaprost (EP(2)), ONO-NT-012 (EP(3)) nor 11-deoxy PGE(1) (EP(3)/EP(4)) showed any protection on the lesions. Indomethacin caused a marked increase in gastric motility; the response preceded the onset of lesions and was inhibited by atropine as well as PGE derivatives acting as EP(1) receptors. Neutrophil chemotaxis was inhibited by PGE(2), butaprost and slightly by 11-deoxy PGE(1), but not by either 17-phenyl PGE(2), ONO-NT-012 or atropine. In addition, indomethacin caused damage similarly in both wild-type and knockout mice lacking EP(1) or EP(3) receptors, yet the protective action of PGE(2) was observed in wild-type and EP(3) receptor knockout mice but totally disappeared in mice lacking EP(1) receptors. CONCLUSION: PGE(2) inhibits indomethacin-induced gastric lesions, through EP(1) receptors, and this effect may be functionally associated with inhibition of gastric motility but not of neutrophil activation/migration.     

Electrophysiological properties of rostral ventrolateral medulla neurons in angiotensin II 1a receptor knockout mice

We compared the electrophysiological properties of neurons in the rostral ventrolateral medulla of neonatal angiotensin II type 1a receptor knockout mice and wild-type mice with responses to angiotensin II, its type-1 receptor blocker candesartan, and its type-2 receptor blocker PD123319. Using the whole-cell patch-clamp technique, we examined the characteristics of rostral ventrolateral medulla neurons in brain stem-spinal cord preparations in which the sympathetic neuronal network is preserved. Baseline membrane potential and firing rate were almost similar between angiotensin II type 1a receptor knockout mice and wild-type mice. Superfusion with angiotensin II depolarized rostral ventrolateral medulla bulbospinal neurons in wild-type mice, whereas it hyperpolarized those in angiotensin II type 1a receptor knockout mice. Because pretreatment with candesartan significantly prevented the angiotensin II-induced depolarization in wild-type mice, the angiotensin II type 1 receptor is crucial for this depolarization. Superfusion with PD123319 depolarized rostral ventrolateral medulla bulbospinal neurons in angiotensin II type 1a receptor knockout mice. PD123319 prevented the angiotensin II-induced hyperpolarization in angiotensin II type 1a receptor knockout mice, and, rather, it induced depolarization. These results suggest that the angiotensin II type 2 receptor in rostral ventrolateral medulla plays an antagonistic role against the angiotensin II type 1a receptor in controlling the neuronal activity of rostral ventrolateral medulla.     

16,16-Dimethyl Prostaglandin E2 Inhibits Indomethacin-Induced Small Intestinal Lesions Through EP3 and EP4 Receptors

We evaluated the effect of various PGE analogs specific to EP receptor subtypes on indomethacin-induced small intestinal lesions in rats and investigated the relationship of EP receptor subtype with the PGE action using EP receptor knockout mice. Animals were administered indomethacin subcutaneously, and they were killed 24 hr later. 16,16-dimethyl prostaglandin E₂ (dmPGE₂) or various EP agonists were administered intravenously 10 min before indomethacin. Indomethacin caused hemorrhagic lesions in the rat small intestine, accompanied with an increase in intestinal motility and the number of enteric bacteria as well as iNOS and MPO activities. Prior administration of dmPGE₂ dose-dependently prevented intestinal lesions, together with inhibition of those functional changes. These effects of dmPGE₂ were mimicked by prostanoids (ONO-NT-012 and ONO-AE1-329), only specific to EP3 or EP4 receptors, although the intestinal motility was inhibited only by ONO-AE1-329. Intestinal mucus secretion and fluid accumulation were decreased by indomethacin but enhanced by dmPGE₂, ONO-NT-012, and ONO-AE1-329 at the doses that prevented intestinal lesions. Indomethacin also caused intestinal lesions in both wild-type and knockout mice lacking EP1 or EP3 receptors, yet the protective action of dmPGE₂ was observed in wild-type and EP1 receptor knockout mice but not the mice lacking EP3 receptors. These results suggest that the intestinal cytoprotective action of PGE₂ against indomethacin is mediated by EP3/EP4 receptors and that this effect is functionally associated with an increase of mucus secretion and enteropooling as well as inhibition of intestinal hypermotility, the former two processes mediated by both EP3 and EP4 receptors, and the latter by EP4 receptors.     

Mechanism by which histamine increases gastric mucosal blood flow in the rat

The mechanism by which histamine increases gastric mucosal blood flow (GMBF) was investigated in the anesthetized rat. The experiment was performed in the presence of tripelennamine, an H₁ antagonist, to focus on the relationship between acid secretion (H₂-receptor-mediated response) and GMBF. The stomach was mounted on a Lucite chamber, perfused with saline, and GMBF was measured by laser Doppler flowmetry simultaneously with acid secretion. Under these conditions, histamine at the submaximal dose significantly increased GMBF as well as acid secretion, and this increase of GMBF was completely blocked when acid secretion was inhibited by cimetidine or omeprazole. The elevation of GMBF caused by histamine was also significantly attenuated when luminal H⁺ was removed by intraluminal perfusion with NaHCO₃ or glycine. Glycine by itself did not affect the increase of acid secretion induced by histamine and the increase of GMBF caused by isoproterenol, yet significantly inhibited the GMBF response induced by pentagastrin. Intraluminal perfusion with HCl also produced an increase of GMBF in a concentration-related manner, even in the presence of omeprazole during histamine infusion. Pretreatment of the animals with indomethacin significantly blocked the GMBF responses induced by either histamine or luminal HCl. These results suggest that the increase of GMBF during acid secretion induced by histamine may be caused by luminal H⁺ and involve endogenous prostaglandins in its mechanism.     

褪黑激素对胃黏膜的保护作用及其机制

目的：探讨褪黑激素和血清素对鼠胃酒精性溃疡及黏膜血流的影响。方法：制备胃在体动物模型。分别皮下注入血清素及同容积生理盐水。用褪黑激素及同容积蒸馏水作为浸育液分别放入各组鼠的胃腔中。30min未毕毕胃黏膜血流后,采用40％的酒精作为浸育液分别放入各组鼠胃腔中,继续检测胃黏膜血流并测量胃黏膜损伤指数。结果：褪黑激素和血清素两者均不能损伤鼠胃黏膜,但血清素可降低胃黏膜血流,且与其剂理相关。褪黑激素可减轻由酒精引起的胃黏膜损伤和胃黏膜血流减少,而血清素可加重酒精引起的胃黏膜损伤和胃黏膜血流减少,但可被褪黑激素部分逆转。结论：胃黏膜的损伤与胃黏膜血流有关,但不是溃疡形成的唯一因素。因此,能拮抗血清的褪黑激素可作为在胃肠道作用的调节剂。     

Modulating Action of Melatonin on Serotonin-Induced Aggravation of Ethanol Ulceration and Changes of Mucosal Blood Flow in Rat Stomachs

Effects of melatonin and serotonin on ethanol ulceration and mucosal blood flow in the rat stomach were investigated. Melatonin and serotonin (5-HT) administration did not produce observable gastric injury in the ex vivo stomach, but the 5-HT dose dependently reduced glandular mucosal blood flow (GMBF) in this organ. Ethanol depressed GMBF and induced visible glandular mucosal injury. The latter effect was prevented by melatonin preincubation. Serotonin pretreatment aggravated the gastric mucosal injury and GMBF changes induced by ethanol; these actions were partially reversed by melatonin. The findings indicate that the GMBF and gastric injury are related; the reduction in FMBF, however, may not be the sole factor responsible for ulceration. The antagonistic effects of melatonin on 5-HT action on the stomach suggest that melatonin may act as a modulator for 5-HT action on the gastrointestinal tract.     

Defining the roles of perforin,Fas/FasL,and tumour necrosis factor alpha in T cell induced mucosal damage in the mouse intestine

BACKGROUND AND AIMS: Mucosal flattening and epithelial cell apoptosis are typical features of T cell induced inflammatory diseases of the bowel, such as coeliac disease and graft versus host disease. Mice injected with a T cell activating anti-CD3 antibody develop a severe diarrhoeal illness. We describe the histological features of this enteropathy and define the effector mechanisms involved in T cell induced mucosal injury in this in vivo model. METHODS: Wild-type and genetically modified mice were injected with the anti-CD3 antibody 3C11 (50 microg). Changes in the murine intestine were characterised by light microscopy analysis and terminal uridine nick-end labelling (TUNEL) assay. The role of perforin, Fas/Fas ligand (FasL), tumour necrosis factor alpha (TNF-alpha), and interferon gamma (IFN-gamma) in T cell induced mucosal damage was assessed using selected immunodeficient mouse strains. RESULTS: T cell activation caused severe damage, including small intestinal mucosal flattening and apoptosis of crypt epithelial cells. Mucosal damage was unaltered in anti-CD3 treated mice lacking IFN-gamma, Fas, or TNF-alpha receptors. In mice lacking TNF-alpha receptors and Fas (TNF-R1xR2 lpr/lpr strain), enterocyte apoptosis was diminished but there was no significant reduction in tissue damage. Apoptosis and mucosal injury were significantly reduced in perforin knockout mice. Abrogation of both FasL and perforin (perforin KOxgld mice) further significantly reduced tissue damage and apoptotic bodies. CONCLUSIONS: T cell induced mucosal injury is mediated by the combined effect of multiple pathways but predominantly by perforin. The redundancy of the mechanisms of tissue damage will have significant impact on therapeutic strategies aimed at specific and targeted inhibition of inflammatory processes.     

No role for prostacyclin IP receptors in duodenal HCO3- secretion induced by mucosal acidification in mice--comparison with capsaicin-induced response

BACKGROUND/AIM: We investigated the role of prostacyclin (PGI2) IP receptors in the acid-induced secretion of HCO3- using IP receptor knockout [IP (-/-)] mice, in comparison with capsaicin-induced secretion. METHODS: Male C57/BL6 mice, both wild-type [WT] and [IP (-/-)], fasted for 18 h were used. Under urethane anesthesia, a proximal duodenal loop was perfused with saline, and the secretion of HCO3- was measured at pH 7.0 using a pH-stat method and by adding 2 mM HCl. The secretion was stimulated by exposure of the loop to 10 mM HCl, capsaicin, PGE2 or cicaprost (a PGI2 agonist) for 10 min. RESULTS: PGE2 stimulated HCO3- secretion in both WT and IP (-/-) mice, while cicaprost increased it in WT but not IP (-/-) mice. Luminal acidification increased the mucosal level of PGE2 as well as 6-keto-PGF1alpha, yet stimulated HCO3- secretion in both WT and IP (-/-) mice, in an indomethacin-inhibitable and sensory neuron-dependent manners. Perfusion of the duodenum with 20 mM HCl for 4 h caused severe damage in WT mice pretreated with indomethacin, but not in control WT or IP (-/-) mice. Capsaicin increased duodenal HCO3- secretion in WT mice, in an indomethacin-sensitive manner, yet no such response was observed in the animals lacking IP receptors. CONCLUSION: The presence of IP receptors is not essential for acid-induced HCO3- secretion and mucosal defense against acid injury in the duodenum, although activation of IP receptors results in stimulation of HCO3- secretion. Although duodenal HCO3- secretion induced by both acid and capsaicin depends on afferent neurons, it seems that the mode of interaction with the afferent neurons differs regarding dependency on the PGI2/IP receptors.     

Nuclear receptors constitutive androstane receptor and pregnane X receptor ameliorate cholestatic liver injury

Cholestasis is associated with accumulation of bile acids and lipids, and liver injury. The constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are xenobiotic nuclear receptors that coordinate protective hepatic responses to potentially toxic stimuli, including bile acids. We investigated the role of these receptors in the regulation of bile acid and lipid metabolism in a bile duct ligation (BDL) model of cholestasis applied to receptor knockout mice. Hepatic damage from bile acid accumulation was increased in both CAR knockout (CARKO) and PXR knockout mice, but bile acid concentrations were lower in CARKO mice. High-density lipoprotein (HDL) cholesterol was elevated in CARKO mice, and serum total cholesterol increased less in CARKO or PXR knockout mice than WT mice after BDL. Gene expression analysis of the BDL knockout animals demonstrated that, in response to cholestasis, PXR and CAR both repressed and induced the specific hepatic membrane transporters Oatp-c (organic anion transporting polypeptide C) and Oatp2 (Na⁺-dependent organic anion transporter 2), respectively. Induction of the xenobiotic transporter multidrug resistance protein 1 in cholestasis was independent of either PXR or CAR, in contrast to the known pattern of induction of multidrug resistance protein 1 by xenobiotics. These results demonstrate that CAR and PXR influence cholesterol metabolism and bile acid synthesis, as well as multiple detoxification pathways, and suggest their potential role as therapeutic targets for the treatment of cholestasis and lipid disorders.     

Impaired duodenal bicarbonate secretion and mucosal integrity in mice lacking prostaglandin E-receptor subtype EP(3).

BACKGROUND & AIMS: To examine the involvement of EP(3) receptors in physiological regulation of duodenal HCO(3)(-) secretion, we disrupted the gene encoding EP receptors in mice by homologous recombination and evaluated acid-induced HCO(3)(-) secretion, which is physiologically important in the mucosal defense against acid injury, using EP(1)- and EP(3)-receptor knockout mice. METHODS: The experiments were performed in the following 3 groups of mice after 18 hours of fasting: wild-type [WT (+/+)] mice, EP(1)-receptor knockout [EP(1) (-/-)] mice, and EP(3)-receptor knockout [EP(3) (-/-)] mice. Under urethane anesthesia, the proximal duodenal loop was perfused with saline that was gassed with 100% O(2), heated at 37 degrees C, and kept in a reservoir, and HCO(3)(-) secretion was measured at pH 7.0 using a pH-stat method and by adding 5 mmol/L HCl. RESULTS: The duodenum of WT (+/+) mice increased HCO(3)(-) secretion in response to luminal perfusion of prostaglandin E(2) and forskolin as well as mucosal acidification. The latter effect was significantly inhibited by prior administration of indomethacin. HCO(3)(-) response to acid was observed in EP(1) (-/-) mice but disappeared totally in EP(3) (-/-) animals, although the acidification increased mucosal PGE(2) generation by similar degrees in all groups. The HCO(3)(-) stimulatory action of PGE(2) was also absent in EP(3) (-/-) but not EP(1) (-/-) mice, but forskolin effect was observed in both groups of animals, similar to WT (+/+) mice. Perfusion of the duodenum with 20 mmol/L HCl for 4 hours caused severe damage in EP(3) (-/-) mice and WT (+/+) animals pretreated with indomethacin, but not in EP(1) (-/-) mice. CONCLUSIONS: The presence of EP(3)-receptors is essential for maintaining duodenal HCO(3)(-) secretion and mucosal integrity against luminal acid.     

Adaptive cytoprotection mediated by prostaglandin I(2) is attributable to sensitization of CRGP-containing sensory nerves

BACKGROUND & AIMS: The phenomenon by which the gastric mucosa is protected in response to mild irritants has been called adaptive cytoprotection, a mechanism believed to be related to production of endogenous prostaglandins (PGs). We tested whether PGs generated by mild irritant prevent injury through the release of calcitonin gene-related peptide (CGRP) from the sensory nerves using prostanoid receptor-knockout mice. METHODS: The stomach was doubly cannulated and perfused with 1 mol/L NaCl or 50% ethanol. CGRP levels in the perfusate were determined by enzyme immunoassay, and the injured area was estimated at the end of perfusion. RESULTS: Preperfusion with mildly hypertonic saline (1 mol/L NaCl) increased generation of gastric PGE(2) and PGI(2) and reduced ethanol-induced mucosal damage. Exposure of ethanol after 1 mol/L NaCl increased intragastric CGRP levels from 166 +/- 27 to 713 +/- 55 pg/2 min (n = 4, P < 0.05), and the protective action of 1 mol/L NaCl was inhibited by indomethacin treatment. CGRP antagonist blocked 1 mol/L NaCl-induced protective effect. Intragastric perfusion of 50% ethanol after administration of PGI(2), but not of PGE(2), increased CGRP levels. Application of 1 mol/L NaCl to IP receptor-knockout mice (IP(-/-)) did not elicit the protective effects seen in the wild-type on ethanol-induced gastric mucosal lesions. Protective effect of 1 mol/L NaCl was observed in EP3 receptor-knockout mice (EP3(-/-)). CGRP level during ethanol perfusion was not increased in IP(-/-) but was increased in EP3(-/-) and wild-type counterparts after preperfusion of 1 mol/L NaCl. CONCLUSIONS: These results indicate that the endogenous PGI(2) generated by 1 mol/L NaCl may have a protective role in gastric mucosal injury through enhancement of CGRP release from gastric mucosa. This mechanism may explain the adaptive cytoprotection observed after treatment with mild irritants.     

Endogenous prostaglandin I2 regulates the neural emergency system through release of calcitonin gene related peptide

BACKGROUND: We previously reported that endogenous prostaglandin I(2), generated by a mild irritant, sensitised calcitonin gene related peptide (CGRP) containing sensory nerves and facilitated the release of CGRP and gastric mucosal protection against ethanol. Administration of capsaicin also inhibited ethanol induced gastric mucosal injury through immediate release of CGRP from primary sensory neurones, which is termed the neural emergency system. In the present study, we tested whether endogenous prostaglandin I(2) also modulates the cytoprotective action of capsaicin using prostaglandin I receptor knockout mice (IP(-/-)). METHODS: The stomachs of IP(-/-) or their wild-type counterparts (IP(+/+)), anaesthetised with urethane (1.225 g/kg), were doubly cannulated from the oesophageal and duodenal sides, and the gastric mucosa was perfused (1 ml/min) with physiological saline. Perfusate was changed to 50% ethanol alone, or 50% ethanol containing capsaicin (16 approximately 1600 micro M). The injured area was estimated at the end of each perfusion experiment. In some animals, CGRP-(8-37), a CGRP antagonist (0.3 mg/kg), or indomethacin (1 mg/kg) was intravenously injected before perfusion of 50% ethanol containing capsaicin. RESULTS: Capsaicin inhibited the injured area in a dose dependent manner. Fifty per cent ethanol containing capsaicin (480 micro M) immediately increased intragastric levels of CGRP although 50% ethanol alone did not. The protective action of capsaicin (480 micro M) against ethanol was completely abolished by intravenous injection of CGRP-(8-37). Indomethacin also inhibited the protective action of capsaicin, and this was accompanied by reduced levels of intragastric CGRP. Intragastric levels of prostaglandin E(2) were not increased by capsaicin treatment but those of 6-keto-prostaglandin F(1alpha), a metabolite of prostaglandin I(2), were markedly increased. No protective action of capsaicin was observed in IP(-/-) which lacked the ability to increase intragastric CGRP levels in response to ethanol containing capsaicin. The CGRP content of the stomach from untreated IP(-/-) did not differ from those in IP(+/+). Capsaicin (160 micro M) together with intragastric perfusion of beraprost sodium (PGI(2) analogue, 2.5 micro g/ml) showed enhanced protection against ethanol induced injury. This enhanced protection was completely blocked by intravenous injection of CGRP-(8-37). CONCLUSIONS: The present results suggest that endogenous prostaglandin I(2) enhances the protective action of the capsaicin mediated neural emergency system against ethanol induced gastric mucosal injury through enhancement of CGRP release.     

Monochloramine impairs mucosal blood flow response and healing of gastric lesions in rats: relation to capsaicin-sensitive sensory neurons

AIMS:We examined the effects of monochloramine (NH2Cl) on the gastric mucosal blood flow (GMBF) response and the healing of ethanol-induced gastric lesions in rats. METHODS: Rats fasted for 18 h were given the 99% ethanol p.o. for induction of gastric lesions, and were fed normally from 1 h later onwards. Monochloramine, at non-ulcerogenic doses (5 to approximately 20 mmol/L), was given p.o. twice daily for 7 days, starting 2 h after ethanol treatment. RESULTS: Gastric lesions caused by ethanol healed almost completely within 7 days with re-epithelialization. The repeated administration of NH2Cl significantly delayed the healing of ethanol-induced gastric lesions in a dose-dependent manner. The damaged mucosa showed a marked rise in H+ permeability, resulting in luminal acid loss, but this process was accompanied by an increase of mucosal blood flow. Monochloramine did not affect the increased mucosal H+ permeability observed in the stomach after damage by ethanol, but significantly inhibited the mucosal hyperemic response associated with luminal acid loss. Prior exposure of the mucosa to NH2Cl (20 mmol/L) did not affect the gastric hyperemic response caused by mucosal application of misoprostol (a prostaglandin E1 derivative) or NOR-3 (a nitric oxide donor), but totally attenuated the increase of GMBF in response to intragastric capsaicin. Impaired healing and GMBF responses were also observed in rats following chemical ablation of capsaicin-sensitive sensory neurons. CONCLUSIONS: These results suggest that NH2Cl impaired the healing of acute gastric mucosal lesions at low concentrations, and this action may be attributable, at least partly, to the impairment of gastric hyperemic response caused by the dysfunction of capsaicin-sensitive sensory neurons.     

The role of prostaglandin E receptor subtypes (EP1, EP2, EP3, and EP4) in bone resorption: an analysis using specific agonists for the respective EPs

PGE2 functions as a potent stimulator of bone resorption. The action of PGE2 is thought to be mediated by some PGE receptor subtypes present in osteoblastic cells. In this study, we examined the involvement of PGE receptor subtypes, EP1, EP2, EP3, and EP4, in PGE2-induced bone resorption using specific agonists for the respective EPs. In mouse calvaria cultures, EP4 agonist markedly stimulated bone resorption, but its maximal stimulation was less than that induced by PGE2. EP2 agonist also stimulated bone resorption, but only slightly. EP1 and EP3 agonists did not stimulate it at all. RT-PCR showed that osteoblastic cells isolated from newborn mouse calvaria expressed all of the EPs messenger RNA (mRNA). Both EP2 agonist and EP4 agonist induced cAMP production and the expression of osteoclast differentiation factor (ODF) mRNA in osteoblastic cells. Simultaneous addition of EP2 and EP4 agonists cooperatively induced cAMP production and ODF mRNA expression. In mouse bone marrow cultures, EP2 and EP4 agonists moderately induced osteoclast formation, but the simultaneous addition of the two agonists cooperatively induced it, similar to that by PGE2. In calvaria culture from EP4 knockout mice, a marked reduction in bone resorption to PGE2 was found. In EP4 knockout mice, EP4 agonist failed to induce bone resorption, but EP2 agonist slightly, but significantly, induced bone resorption. These findings suggest that PGE2 stimulates bone resorption by a mechanism involving cAMP and ODF, which is mediated mainly by EP4 and partially by EP2.