The role of l-cysteine/Hydrogen sulfide pathway on ß3-Adrenoceptor- induced relaxation in mouse gastric fundus.

In this study, we investigated the possible role of the l-cysteine/hydrogen sulfide pathway in ß3-adrenoceptors-mediated relaxation in isolated mouse gastric fundus tissue. l-cysteine (endogenous H2S; 10-6-10-2 M), sodium hydrogen sulfide (NaHS; exogenous H2S; 10-6-10-3 M), selective ß3-adrenoceptors agonist BRL 37344 (10-9-10-4 M) and non-selective ß-adrenoceptor agonist isoprenaline (10-9-10-4 M) produced concentration-dependent relaxation in mouse gastric fundus. The non-selective ß-adrenoceptors antagonist propranolol (10-6 M) inhibited the relaxant response to isoprenaline but not to BRL 37344. On the other hand, the selective ß3-adrenoceptors antagonist SR 59230A (10-5 M) inhibited the relaxant responses to BRL 37344. In addition, cystathionine-gamma-lyase (CSE) inhibitor D,L-propargylglycine (PAG, 10-2 M), cystathionine-beta-synthase inhibitor (CBS) aminooxyacetic acid (AOAA, 10-2 M), and the combination of these inhibitors significantly reduced the relaxant responses induced by l-cysteine and BRL 37344. Pre-incubation of gastric fundal strips with propranolol (10-6 M) and SR 59230A (10-5 M) did not affect relaxations to l-cysteine and NaHS. Also, the existence of CSE, CBS, 3-mercaptopurivate sulfur transferase (3-MST) enzymes and ß3-adrenoceptors were detected in gastric fundal tissue. Furthermore, basal H2S release was detected in the measurements. H2S level increased in the presence of l-cysteine, NaHS, and BRL 37344. The increase in H2S level by l-cysteine and BRL 37344 decreased significantly with PAG and AOAA enzyme inhibitors. These results suggest that endogenous H2S is synthesized from l-cysteine at least by CBS and CSE enzymes. Also, ß3-adrenoceptors are found in the mouse stomach fundus and mediate BRL 37344-induced relaxations, and l-cysteine/hydrogen sulfide pathway plays a partial role in ß3-adrenoceptors-mediated relaxation in mouse gastric fundus tissue.

H2S, a novel gasotransmitter, involves in gastric accommodation.

H2S is produced mainly by two enzymes:cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE), using L-cysteine (L-Cys) as the substrate. In this study, we investigated the role of H2S in gastric accommodation using CBS(+/-) mice, immunohistochemistry, immunoblot, methylene blue assay, intragastric pressure (IGP) recording and electrical field stimulation (EFS). Mouse gastric fundus expressed H2S-generating enzymes (CBS and CSE) and generated detectable amounts of H2S. The H2S donor, NaHS or L-Cys, caused a relaxation in either gastric fundus or body. The gastric compliance was significantly increased in the presence of L-Cys (1 mM). On the contrary, AOAA, an inhibitor for CBS, largely inhibited gastric compliance. Consistently, CBS(+/-) mice shows a lower gastric compliance. However, PAG, a CSE inhibitor, had no effect on gastric compliances. L-Cys enhances the non-adrenergic, non-cholinergic (NANC) relaxation of fundus strips, but AOAA reduces the magnitude of relaxations to EFS. Notably, the expression level of CBS but not CSE protein was elevated after feeding. Consistently, the production of H2S was also increased after feeding in mice gastric fundus. In addition, AOAA largely reduced food intake and body weight in mice. Furthermore, a metabolic aberration of H2S was found in patients with functional dyspepsia (FD). In conclusion, endogenous H2S, a novel gasotransmitter, involves in gastric accommodation.

Tonic and phasic smooth muscle contraction is not regulated by the PKCα - CPI-17 pathway in swine stomach antrum and fundus.

Regulation of myosin light chain phosphatase (MLCP) via protein kinase C (PKC) and the 17 kDa PKC-potentiated inhibitor of myosin light chain phosphatase (CPI-17) has been reported as a Ca(2+) sensitization signaling pathway in smooth muscle (SM), and thus may be involved in tonic vs. phasic contractions. This study examined the protein expression and spatial-temporal distribution of PKCα and CPI-17 in intact SM tissues. KCl or carbachol (CCh) stimulation of tonic stomach fundus SM generates a sustained contraction while the phasic stomach antrum generates a transient contraction. In addition, the tonic fundus generates greater relative force than phasic antrum with 1 µM phorbol 12, 13-dibutyrate (PDBu) stimulation which is reported to activate the PKCα - CPI-17 pathway. Western blot analyses demonstrated that this contractile difference was not caused by a difference in the protein expression of PKCα or CPI-17 between these two tissues. Immunohistochemical results show that the distribution of PKCα in the longitudinal and circular layers of the fundus and antrum do not differ, being predominantly localized near the SM cell plasma membrane. Stimulation of either tissue with 1 µM PDBu or 1 µM CCh does not alter this peripheral PKCα distribution. There are no differences between these two tissues for the CPI-17 distribution, but unlike the PKCα distribution, CPI-17 appears to be diffusely distributed throughout the cytoplasm under relaxed tissue conditions but shifts to a primarily peripheral distribution at the plasma membrane with stimulation of the tissues with 1 µM PDBu or 1 µM CCh. Results from double labeling show that neither PKCα nor CPI-17 co-localize at the adherens junction (vinculin/talin) at the membrane but they do co-localize with each other and with caveoli (caveolin) at the membrane. This lack of difference suggests that the PKCα - CPI-17 pathway is not responsible for the tonic vs. phasic contractions observed in stomach fundus and antrum.

Differential expression of multidrug resistance protein 5 and phosphodiesterase 5 and regulation of cGMP levels in phasic and tonic smooth muscle.

Previous studies have identified differences in the expression of proteins that regulate myosin light chain phosphorylation and contraction in tonic and phasic smooth muscle. cGMP plays a critical role in smooth muscle relaxation and is important for optimal function of phasic and tonic smooth muscle. The intracellular cGMP levels are regulated by its hydrolysis via phosphodiesterase 5 (PDE5) and efflux via novel multidrug resistance protein 5 (MRP5). In the present study we tested the hypothesis that the differences in the phasic and tonic behavior of smooth muscles may be related to differences in mechanisms that terminate cGMP signaling. Expression of PDE5 and MRP5 was significantly (more than 2-fold) higher in fundus compared with antrum. The NO donor S-nitrosoglutathione (GSNO) caused an increase in PDE5 activity and intra- and extracellular cGMP levels in both fundus and antrum. Stimulation of PDE5 activity and increase in extracellular cGMP were significantly higher in fundus, whereas increase in intracellular cGMP was significantly higher in antrum. GSNO-induced increase in extracellular cGMP was blocked in dispersed cells by the cyclic nucleotide export blocker probenecid and in cultured muscle cells by depletion of ATP or suppression of MRP5 by siRNA, providing evidence that cGMP efflux was mediated by ATP-dependent export via MRP5. Consistent with the higher expression and activity levels of PDE5 and MRP5, GSNO-induced PKG activity and muscle relaxation were significantly lower in muscle cells from fundus compared with antrum. Thus higher expression of PDE5 and MRP5 in muscle cells from fundus correlates with tonic phenotype of muscle.

CDP-choline-induced contractions in the mouse gastric fundus through purinoceptors and Rho/Rho-kinase signalling.

AIMS: This study aimed to investigate the effects of cytidine-5'-diphosphocholine (CDP-choline), an endogenous lipid precursor, on the reactivity of the mouse gastric fundus and to determine the mechanism(s) mediating its effects. MAIN METHODS: Possible contractile effect of CDP-choline (10(-5)-10(-2)M) was investigated in the absence and presence of a muscarinic receptor antagonist, atropine (3 × 10(-6)M), an acetylcholine esterase inhibitor, physostigmine (10(-6)M), a Na(+) channel blocker, tetrodotoxin (TTX, 3 × 10(-6)M), a Rho-kinase inhibitor, Y-27632 (10(-5) M), a purinoceptor antagonist, suramin (2 × 10(-4)M), a nitric oxide synthase inhibitor, N(G)-nitro-L-arginine (L-NA, 3 × 10(-4)M), a Ca(2+) channel blocker, nifedipine (10(-6)M), an α(7) nicotinic receptor antagonist, methyllycaconitine citrate (MLA, 10(-6)M) and a G protein (G(i/o)) inhibitor, pertussis toxin (PTX, 2 µg/ml). The metabolites of CDP-choline, namely choline (10(-4)-10(-2)M), cytidine 5'-triphosphate (CTP, 10(-5)-10(-2)M), cytidine (10(-5)-10(-2)M) and cytidine monophosphate (CMP, 10(-3)-10(-2)M) were also tested. Besides, phosphorylation of MYPT1, which indicates Rho-kinase activity, was also detected. KEY FINDINGS: CDP-choline produced contractions in a concentration-dependent manner. The contractions were not affected by atropine, physostigmine, TTX, PTX, MLA or L-NA. However, Y-27632, suramin or nifedipine partly reduced these contractions. CDP-choline increased phosphorylation of MYPT1. Among CDP-choline metabolites, cytidine had no contractile effects. However, choline induced considerable contractions, which were sensitive to atropine. CMP and CTP had also contractile activity, comparable to that of CDP-choline. SIGNIFICANCE: These results suggest that CDP-choline produced contraction through, at least in part, purinoceptors and Rho/Rho-kinase signalling in the mouse gastric fundus.

Self-renewal of the human gastric epithelium: new insights from expression profiling using laser microdissection.

The gastric mucosa is subject to continual bidirectional renewal by differentiation from stem and transit amplifying cells. It was the aim of this study to characterize the self-renewal of the human gastric mucosa and its two major types of glands in the fundus and antrum, respectively. Three characteristic regions (pit, proliferative, and lower neck regions) were isolated from fundic and antral units by the use of laser microdissection, and expression profiles concerning 15 marker genes were generated by RT-PCR analysis. The surface mucous cells (SMCs) of fundic and antral units differed in their expression of at least four secretory genes, i.e., gastric lipase, TFF3, FCGBP, and lysozyme. The maturation of mucous neck cells was shown to occur stepwise, first towards a mucous phenotype followed by a serous differentiation step. Also, a stepwise maturation of both the antral SMCs and antral gland cells was observed. Additionally, the presence of gastric lipase was also demonstrated for the first time in antral gland cells. In conclusion, the different expression profiles of SMCs of the fundic and antral units could be the basis for the different self-renewal rates of fundic and antral SMCs and could influence the spatial organization of the bacterial microbiota within the various parts of the gastric mucosa.

Lysozyme overexpression in fundic gland polyps.

UNLABELLED: Backgroud: Helicobacter pylori (Hp) rarely proliferates in patients with fundic gland polyps (FGPs). We recently found that FGPs express lysozyme, one of the natural defence substances against infection. We aimed to assess the degree of lysozyme expression in a cohort of consecutive FGPs. MATERIALS AND METHODS: A total of 153 gastric biopsies were investigated: 93 with FGPs, 30 with normal mucosa (Nm), 15 with Hp-induced chronic gastritis (Hp-gastritis) and 15 with chronic gastritis without Hp infection (non-Hp-gastritis). Sections were stained with anti-lysozyme (muramidase). RESULTS: Lysozyme was slightly to moderately expressed in the surface and foveolar pits, being markedly expressed in the neck glands in Nm, in non-Hp and Hp-gastritis. The ratio of lysozyme neck glands-foveoli was higher in non-Hp than in Nm and even higher in Hp-gastritis. In FGPs, lysozyme was markedly expressed in the surface, the foveolar pits and the cells that partly or entirely covered the microcysts. DISCUSSION AND CONCLUSION: While the moderate expansion of the lysozyme-producing cells of the neck glands in Hp-gastritis might be insufficient to eradicate these bacteria, the overproduction of lysozyme in the epithelium covering FGP could be an explanation for the lack of Hp proliferation in these patients.

GERD is associated with shortened telomeres in the squamous epithelium of the distal esophagus.

Telomeres are repetitive DNA sequences located at the ends of chromosomes. Telomeres are shortened by repeated cell divisions and by oxidative DNA damage, and cells with critically shortened telomeres cannot divide. We hypothesized that chronic gastroesophageal reflux disease (GERD)-induced injury of the esophageal squamous epithelium results in progressive telomeric shortening that eventually might interfere with mucosal healing. To address our hypothesis, we compared telomere length and telomerase activity in biopsy specimens of esophageal squamous epithelium from GERD patients and control patients. Endoscopic biopsies were taken from the esophageal squamous epithelium of 38 patients with GERD [10 long-segment Barrett's esophagus (LSBE), 15 short-segment (SSBE), 13 GERD without Barrett's esophagus] and 16 control patients without GERD. Telomere length was assessed using the terminal restriction fragment assay, and telomerase activity was studied by the PCR-based telomeric repeat amplification protocol assay. Patients with GERD had significantly shorter telomeres in the distal esophagus than controls [8.3 +/- 0.5 vs. 10.9 +/- 1.5 (SE) Kbp, P = 0.043]. Among the patients with GERD, telomere length in the distal esophagus did not differ significantly in those with and without Barrett's esophagus (LSBE 7.9 +/- 0.8, SSBE 8.6 +/- 0.9, GERD without BE 8.7 +/- 1.0 Kbp). No significant differences in telomerase activity in the distal esophagus were noted between patients with GERD and controls (4.0 +/- 0.39 vs. 5.2 +/- 0.53 RIUs). Telomeres in the squamous epithelium of the distal esophagus of patients who have GERD, with and without Barrett's esophagus, are significantly shorter than those of patients without GERD despite similar levels of telomerase activity.

Gastric graft-versus-host disease revisited: does proton pump inhibitor therapy affect endoscopic gastric biopsy interpretation?

Accurate diagnosis of gastrointestinal graft-versus-host disease (GvHD) is important, as it contributes significantly to postallogeneic stem cell transplant (SCT) morbidity and mortality. To test the hypothesis that proton pump inhibitor (PPI) therapy may interfere with histologic evaluation of gastric GvHD by inducing apoptosis, we evaluated epithelial apoptotic body counts in antral and fundic biopsies from SCT recipients and control patients, both taking and not taking PPIs at the time of endoscopic biopsy. Hematoxylin and eosin-stained slides of gastric biopsies from 130 patients (75 allogeneic SCT with GvHD on clinical and histologic grounds, and a comparison group of 55 age- and sex-matched nontransplant patients with histologically normal gastric biopsies) were reviewed. The groups were further stratified into patients taking (PPI+) and not taking PPIs (PPI-) at the time of biopsy. Apoptotic bodies (AB)/10 (400 x) high power fields (HPF) were quantified for each case. Mean apoptotic body counts were then calculated for each case group. Seventy antral cases (31 control and 39 transplant) were also evaluated via gastrin immunohistochemistry, and the mean number of gastrin positive cells/400 x HPF calculated. In the PPI- groups, apoptosis was increased in biopsies from transplant patients, compared with controls, both in antral and fundic mucosa. In PPI+ patients, there was significantly more apoptosis in the gastric body in transplant patients than in controls. However, comparing antral biopsies from control and transplant PPI+ patients, there was no significant difference in AB quantitation. More apoptosis was seen in antral biopsies from PPI+ control patients when compared with PPI- control patients (P = 0.009). Mean numbers of gastrin positive cells/400 x HPF were increased in both control and transplant patients taking PPIs (85 and 58, respectively) compared with samples from those patients not taking PPIs (48 and 51, respectively). PPI therapy is associated with increased apoptosis in antral biopsies and may interfere with the evaluation of GvHD in biopsies from this site. A similar increase in apoptosis was not seen in fundic biopsies; biopsy of the gastric fundus rather than antrum may be preferable for the diagnosis of upper gastrointestinal GvHD.

Influence of relaxin on the neurally induced relaxant responses of the mouse gastric fundus.

The peptide hormone relaxin has been reported to depress the amplitude of contractile responses in the mouse gastric fundus by upregulating nitric oxide (NO) biosynthesis at the neural level. In the present study, we investigated whether relaxin also influenced nonadrenergic, noncholinergic (NANC) gastric relaxant responses in mice. Female mice in proestrus or estrus were treated for 18 h with relaxin (1 microg s.c.) or vehicle (controls). Mechanical responses of gastric fundal strips were recorded via force-displacement transducers. In carbachol precontracted strips from control mice and in the presence of guanethidine, electrical field stimulation (EFS) elicited fast relaxant responses that may be followed by a sustained relaxation. All relaxant responses were abolished by tetrodotoxin. Relaxin increased the amplitude of the EFS-induced fast relaxation without affecting either the sustained one or the direct smooth muscle response to papaverine. In the presence of the NO synthesis inhibitor L-N(G)-nitro arginine (L-NNA), that abolished the EFS-induced fast relaxation without influencing the sustained one, relaxin was ineffective. In strips from relaxin-pretreated mice, EFS-induced fast relaxations were enhanced in amplitude with respect to the controls, while sustained ones as well as direct smooth muscle responses to papaverine were not changed. Further addition of relaxin to the bath medium did not influence neurally induced fast relaxant responses, whereas L-NNA did. In conclusion, in the mouse gastric fundus, relaxin enhances the neurally induced nitrergic relaxant responses acting at the neural level.

Nitrergic relaxation in rat gastric fundus: influence of mechanism of induced tone and possible role of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase.

The aim of this study was to investigate the influence of the mechanism of induced tone and the role of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) in nitrergic relaxation of rat gastric fundus. Prostaglandin F(2alpha) (PGF(2alpha)), thapsigargin (TSG) and cyclopiazonic acid (CPA) were used in concentrations that induced a similar contraction (20 g force/g tissue). Nifedipine (3 x 10(-7) M) completely relaxed PGF(2alpha)-contracted tissues and relaxed tissues contracted by TSG and CPA by 20 +/- 6% and 56 +/- 12% respectively; contraction induced by the three contractile agents was fully reversed by a general Ca2+ entry blocker 1-[2-(4-methoxyphenyl)-2-[3-(4-metoxyphenyl)propoxy]ethyl-1H-imidazole HCl (SKF 96365; 10(-5) M). In the presence of nifedipine (3 x 10(-7) M) or verapamil (10(-5) M), PGF(2alpha) and CPA-induced contractions were still approximately 50% relaxed by SKF 96365. This suggests that contractions induced by PGF(2alpha) are related to Ca2+ entry through L-type voltage-operated Ca2+ channels and that contractions by TSG are mainly related to Ca2+ entry through store-operated Ca2+ channels. Relaxant responses to exogenous nitric oxide (NO), to endogenous NO released by electrical field stimulation, and to vasoactive intestinal polypeptide (VIP) were studied in tissues contracted by TSG and CPA and compared to responses in tissues contracted by PGF(2alpha). Responses to exogenous and endogenous NO were greatly attenuated in TSG-contracted tissues, but not in CPA-contracted tissues. When contraction was induced by CPA in the presence of nifedipine or verapamil, relaxations to exogenous and endogenous NO were also significantly reduced. Relaxation induced by VIP was reduced in tissues contracted by either TSG or CPA in the presence of nifedipine or verapamil. These results suggest that the ability of the nitrergic neurotransmitter to induce relaxation of rat gastric fundus is influenced by the mechanism used to induce tone and are indicative for a role for SERCA in nitrergic relaxation. However, activation of SERCA appears to not be unique for nitrergic relaxation, but might also be used by VIP, a co-transmitter of NO in this tissue.

Decreased UCP2 mRNA expression in rat stomach following vagotomy: novel role for UCP2 as free radical scavenger in the stomach?

Uncoupling protein 2 (UCP2) is a protein, located in the inner mitochondrial membrane, which dissipates the proton gradient of this membrane and uncouples respiration from oxidative phosphorylation. We found, by in situ hybridisation, UCP2 mRNA to be located in the proliferating zone of the mucous neck cells in the fundus part of the rat stomach. We also found that UCP2 expression in fundus was significantly decreased after seven days of vagotomy. Furthermore, we found manganese-containing superoxide dismutase (SOD2), in fundus, to be down-regulated in a way similar to UCP2. The amount of ATP was significantly decreased following vagotomy. It is concluded that UCP2 in the gastro-intestinal tract is regulated through vagal innervation and suggested to act as a free radical scavenger.

Regulation of amylin release from cultured rabbit gastric fundic mucosal cells.

BACKGROUND: Amylin (islet amyloid polypeptide) is a hormone with suggested roles in the regulation of glucose homeostasis, gastric motor and secretory function and gastroprotection. In the gastric mucosa amylin is found co-localised with somatostatin in D-cells. The factors regulating gastric amylin release are unknown. In this study we have investigated the regulation of amylin release from gastric mucosal cells in primary culture. Rabbit fundic mucosal cells enriched for D-cells by counterflow elutriation were cultured for 40 hours. Amylin and somatostatin release over 2 hours in response to agonists were assessed. RESULTS: Amylin release was significantly enhanced by activation of protein kinase C with phorbol-12-myristate-13-acetate, adenylate cyclase with forskolin and elevation of intracellular calcium with A23187. Cholecystokinin (CCK), epinephrine and glucagon-like peptide-1 (GLP-1) each stimulated amylin release in a dose-dependent manner. Maximal CCK-stimulated release was greater than either epinephrine or GLP-1, even when the effects of the latter two were enhanced by isobutylmethylxanthine. Stimulated amylin release was significantly inhibited by carbachol (by 51-59%) and octreotide (by 33-42%). Somatostatin release paralleled that of amylin. CONCLUSIONS: The cultured D-cell model provides a means of studying amylin release. Amylin secretion is stimulated by receptor-dependent and -independent activation of Ca2+/protein kinase C and adenylate cyclase pathways. Inhibition involves activation of muscarinic receptors and auto-regulation by somatostatin.

Investigation of the potential modulatory effect of biliverdin, carbon monoxide and bilirubin on nitrergic neurotransmission in the pig gastric fundus.

In porcine gastric fundus, we have investigated the colocalization of the bile pigment biosynthetic enzymes heme oxygenase-2 and biliverdin reductase with neuronal nitric oxide synthase (nNOS), the effect of carbon monoxide (CO) on fundic circular smooth muscle and the possible modulatory effect of the bile pigments biliverdin and bilirubin on CO-mediated relaxations and on nitrergic relaxation. Heme oxygenase-2 and biliverdin reductase immunoreactivity was present in all nNOS containing myenteric neurons. CO induced a concentration-dependent relaxation of fundic circular smooth muscle strips, which was completely blocked by the specific guanylate cyclase inhibitor 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ). 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), biliverdin and bilirubin strongly enhanced the amplitude of the CO-induced relaxation. Tin protoporphyrin had no effect on electrically induced nitrergic relaxation, but spectrophotometric analysis learned that incubation of porcine gastric fundus circular muscle strips with tin protoporphyrin did not influence heme oxygenase activity. In conclusion, our data suggest that nitrergic neurons in the pig gastric fundus are able to produce biliverdin and bilirubin, and that these agents potentiate the relaxant effect of CO, which is formed concomitantly with biliverdin by heme oxygenase-2.

L-citrulline recycling by argininosuccinate synthetase and lyase in rat gastric fundus.

The aim of this study was to investigate in rat gastric fundus whether L-citrulline, the co-product in the nitric oxide (NO) biosynthesis catalyzed by neuronal nitric oxide synthase (nNOS), can be converted back to the nNOS substrate L-arginine. Immunohistochemistry showed that argininosuccinate synthetase and argininosuccinate lyase, that mediate transformation of L-citrulline to L-arginine in the ureum cycle in hepatocytes, co-localize with nNOS. In longitudinal smooth muscle strips, L-arginine as well as L-citrulline (10(-3) M) was capable of completely respectively partially preventing the N(G)-nitro-L-arginine methyl ester (L-NAME) (3 x 10(-5) M)-induced inhibition of electrically induced nitrergic relaxations, whereas D-citrulline (10(-3) M) was not. The L-citrulline-mediated prevention of the L-NAME-induced inhibition was reduced by L-glutamine (3 x 10(-3) M), the putative L-citrulline uptake inhibitor, and by succinate, an argininosuccinate lyase inhibitor. The results demonstrate that the L-citrulline recycling mechanism is active in rat gastric fundus. Recycling of L-citrulline might play a role in providing sufficient amounts of nNOS substrate during long-lasting relaxations in gastric fundus after food intake.

RhoA kinase and protein kinase C participate in regulation of rabbit stomach fundus smooth muscle contraction.

1. The degree to which the RhoA kinase (ROK) blockers, Y-27632 (1 micro M) and HA-1077 (10 micro M), and the PKC blocker, GF-109203X (1 micro M), reduced force produced by carbachol, a muscarinic receptor agonist, and phenylephrine, an alpha-adrenoceptor agonist, was examined in rabbit stomach fundus smooth muscle. 2. When examining the effect on cumulative carbachol concentration-response curves (CRCs), ROK and PKC blockers shifted the potency EC50 to the right but did not reduce the maximum response. 3. In a single-dose carbachol protocol using moderate ( approximately EC50 and maximum carbachol concentrations, Y-27632 and HA-1077 reduced peak force, but GF-109203X had no effect. By contrast, all three agents inhibited the carbachol contractions of rabbit bladder (detrusor) smooth muscle. 4. Compared to carbachol, phenylephrine produced a weaker maximum response that was not inhibited by phentolamine, atropine nor capsaicin but was inhibited by Y-27632, HA-1077 and GF-109203X. 5. In detrusor, classical down-regulation occurred, but in fundus, up-regulation of responsiveness occurred. This up-regulation in fundus may have been a post-receptor event, because a KCl-induced contraction produced after a carbachol CRC was stronger than one produced before the carbachol stimulus. 6. In conclusion, these data suggest that ROK plays a critical role in the regulation of rabbit fundus smooth muscle contraction, which is distinct from chicken gizzard smooth muscle, where ROK is reported to exist but to not play a role in muscarinic receptor-induced contraction. Additional unique findings are that PKC participates in phenylephrine- but not carbachol-induced contraction in fundus, that carbachol does not activate identical subcellular signalling systems in fundus and detrusor, and that fundus, unlike detrusor, responds to carbachol stimulation with post-receptor up-regulation of contraction.

Differential autophosphorylation of CaM kinase II from phasic and tonic smooth muscle tissues.

Ca+/calmodulin-dependent protein kinase II (CaM kinase II) is regulated by calcium oscillations, autophosphorylation, and its subunit composition. All four subunit isoforms were detected in gastric fundus and proximal colon smooth muscles by RT-PCR, but only the gamma and delta isoforms are expressed in myocytes. Relative gamma and delta message levels were quantitated by real-time PCR. CaM kinase II protein and Ca2+/calmodulin-stimulated (total) activity levels are higher in proximal colon smooth muscle lysates than in fundus lysates, but Ca2+/calmodulin-independent (autonomous) activity is higher in fundus lysates. CaM kinase II in fundus lysates is relatively unresponsive to Ca2+/calmodulin. Alkaline phosphatase decreased CaM kinase II autonomous activity in fundus lysates and restored its responsiveness to Ca2+/calmodulin. Acetylcholine (ACh) increased autonomous CaM kinase II activity in fundus and proximal colon smooth muscles in a time- and dose-dependent manner. KN-93 enhanced ACh-induced fundus contractions but inhibited proximal colon contractions. The different properties of CaM kinase II from fundus and proximal colon smooth muscles suggest differential regulation of its autophosphorylation and activity in tonic and phasic gastrointestinal smooth muscles.

Induction of heme oxygenase in guinea-pig stomach: roles in contraction and in single muscle cell ionic currents.

The role of heme oxygenase reaction products in modulation of stomach fundus excitability was studied. The presence of constitutive heme oxygenase 2 was verified in myenteric ganglia by immunohistochemistry. The role of inducible heme oxygenase isoenzyme was investigated after invivo treatment of animals with CoCl2 (80 mg kg-1 b.w) injected subcutaneously 24 h before they were killed. This treatment resulted in increased production of bilirubin and positive staining for the inducible isoform in stomach smooth muscle and vast induction in the liver. In both control and treated animals haemin, applied to the bath as a substrate of heme oxygenase caused significant decrease of prostaglandin F2alpha-induced tone, and ameliorated the relaxatory response of the fundic strips to electrical field stimulation. Both effects were antagonized by Sn-protoporphyrin IX, competitive heme oxygenase inhibitor, and were found to be neuronally dependent. In single freshly isolated smooth muscle cells from control animals haemin caused a concentration-dependent increase of the whole cell K+ currents, which was not affected by Sn-protoporphyrin IX, cyclic guanosine monophosphate (cGMP)-dependent protein kinase or guanylyl cyclase antagonists, but was reversed by various antioxidants and abolished by an NO scavenger. In cells from treated animals the K+ current increasing effect of haemin did not depend on the presence of antioxidants, but was abolished by protein kinase G and guanylyl cyclase inhibitors, depletors of intracellular Ca2+ pools or Sn-protoporphyrin IX. Biliverdin did not affect contraction or ionic currents. Thus, this is the first study demonstrating that heme oxygenase is an inducible enzyme in guinea-pigs, which exerts a modulatory role on gastric smooth muscle excitability via carbon monoxide production.

Immunohistochemical localization of the antioxidant enzymes biliverdin reductase and heme oxygenase-2 in human and pig gastric fundus.

The intrinsic antioxidant capacities of the bile pigments biliverdin and bilirubin are increasingly recognized since both heme degradation products can exert beneficial cytoprotective effects due to their scavenging of oxygen free radicals and interaction with antioxidant vitamins. Several studies have been published on the localization of the carbon monoxide producing enzyme heme oxygenase-2 (HO-2), which concomitantly generates biliverdin; histochemical data on the distribution of biliverdin reductase (BVR), converting biliverdin to bilirubin, are still very scarce in large mammals including humans. The present study revealed by means of immunohistochemistry the presence of BVR and HO-2 in mucosal epithelial cells and in the endothelium of intramural vessels of both human and porcine gastric fundus. In addition, co-labeling with the specific neural marker protein-gene product 9.5 (PGP 9.5) demonstrated that both BVR and HO-2 were present in all intrinsic nerve cell bodies of both submucous and myenteric plexuses, while double labeling with c-Kit antibody confirmed their presence in intramuscular interstitial cells of Cajal (ICC). Our results substantiate the hypothesis that BVR, through the production of the potent antioxidant bilirubin, might be an essential component of normal physiologic gastrointestinal defense in man and pig.

Effect of 2,4,6-trinitrobenzenesulphonic acid (TNBS)-induced ileitis on the motor function of non-inflamed rat gastric fundus.

During intestinal inflammation, motility disturbances are not restricted to inflamed regions, but may also occur in remote non-inflamed sites of the gastrointestinal tract. Our aim was to investigate the motor function of the gastric fundus after the induction of terminal ileitis in the rat. Ileal inflammation was induced by intraluminal installation of 2,4,6-trinitrobenzenesulphonic acid (TNBS) into the ileum. Inflammation was assessed both histologically and biochemically. Contractions and relaxations of longitudinal muscle strips from the gastric fundus were studied 36 h and 1 week later. During the acute phase of ileal inflammation (36 h), the non-inflamed stomach was distended. The contractility of longitudinal muscle strips of the gastric fundus was decreased due to a post-receptor defect. In addition, nonadrenergic noncholinergic (NANC) relaxations were inhibited due to neuronal dysfunction. Aortic contractility remained normal and the mere presence of food in the stomach did not account for the disturbed neuromuscular function in the gastric fundus. Ablation of extrinsic primary afferent neurones by capsaicin further impaired gastric fundus contractility. Transection and re-anastomosis of the jejunum reversed the effect of TNBS-induced ileitis on the neuromuscular function of the gastric fundus. One week after TNBS, cholinergic neurotransmission was increased in the gastric fundus. During acute ileitis, smooth muscle cell contractility and inhibitory NANC neurotransmission are inhibited in the non-inflamed gastric fundus. This phenomenon may be mediated by intrinsic connections within the enteric nervous system.