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Biomedical subjects

K Takeuchi

Publications and source records attributed to K Takeuchi.

At least 487 records · Page 27Linked to original sources
PubMed1997

Effects of selective cyclooxygenase-2 inhibitors on alkaline secretory and mucosal ulcerogenic responses in rat duodenum.

Effects of the selective cyclooxygenase-2 (COX-2) inhibitors such as NS-398 and nimesulide on duodenal HCO3- secretory and ulcerogenic responses to mucosal acidification were examined in rats, in comparison with indomethacin, a nonselective COX inhibitor. Duodenal HCO3- secretion in anesthetized rats was increased in response to mucosal acidification. The increased HCO3- response to acid was significantly suppressed by pretreatment with indomethacin (10 mg kg(-1), s.c.), while both NS-398 and nimesulide (10 mg kg(-1), s.c.) had no effect on this response. The luminal release of prostaglandin E2 (PGE2) was increased during and after mucosal acidification, and this response was significantly inhibited by indomethacin but not NS-398 or nimesulide. Indomethacin provoked hemorrhagic lesions in the duodenum when acid hypersecretion was concomitantly induced by histamine (8 mg kg(-1) hr(-1), i.v.), while either NS-398 or nimesulide did not cause damage in the duodenum. Either of these drugs had no effect on histamine-induced acid secretion. On the other hand, both NS-398 and nimesulide showed a significant suppression against carrageenan-induced rat paw edema, similar to indomethacin. The present study supports a mediator role for endogenous PGs in duodenal HCO3- secretion in response to mucosal acidification and suggests that COX-1 but not COX-2 is a key enzyme in regulating this process and maintaining the mucosal integrity against acid in the duodenum.

Animals↗
PubMed1997

Rat kidney thromboxane synthase: cDNA cloning and gene expression regulation in hydronephrotic kidney.

We isolated a rat homolog of thromboxane (TX) synthase cDNA (-1.8 kb) from the kidney with a fragment of human TX synthase cDNA amplified by polymerase chain reaction with placenta cDNA as a template. Northern blot analysis has shown that rat TX synthase gene is expressed abundantly in lung, liver, and uterus; moderately in kidney. TX synthase mRNA expression was up-regulated in hydronephrotic kidney made by ureter ligation. In conclusion, we have revealed the structure of rat kidney TX synthase. Up-regulation of renal TX synthase, which may cause stimulation of TX synthesis, is possibly implicated in the tissue injury in hydronephrotic kidney.

Amino Acid Sequence↗
PubMed1997

Nitric oxide, prostaglandin, and sensory neurons in gastric mucosal blood flow response during acid secretion in rats.

1. The mechanism underlying the increase of gastric mucosal blood flow (GMBF) during acid secretion induced by pentagastrin was investigated in anesthetized rats, in relation to nitric oxide (NO), prostaglandin (PG), and sensory neurons. 2. An intravenous infusion of pentagastrin at 60 micrograms/kg/h (submaximal dose) produced an increase of acid secretion and GMBF as determined by laser Doppler flowmetry, and the GMBF response was totally attenuated when the acid secretion was inhibited by omeprazole or when the luminal H+ was removed by mucosal perfusion with glycine (200 mM). 3. Prior administration of NG-nitro-L-arginine methyl ester (L-NAME, 5 mg/kg, IV), a NO synthase inhibitor, significantly mitigated the GMBF response to pentagastrin, without any influence on acid secretion, and this effect was antagonized by coadministration of L-arginine (500 mg/kg IP). 4. The increase of GMBF during pentagastrin infusion also was significantly mitigated by indomethyacin (5 mg/kg, SC) or sensory deafferentation following capsaicin pretreatment, had no effect on the acid secretion, and was totally inhibited by the combined treatments with indomethacin plus L-NAME in addition to sensory deafferentation. 5. Pentagastrin infusion for 8 hr did not by itself cause any macroscopic damage in the stomach, but additional treatments with L-NAME, and indomethacin plus sensory deafferentation provoked severe lesions in the gastric mucosa. 6. These results suggest that the increase of GMBF induced by submaximal dose of pentagastrin totally depends on luminal H+. This process seems to be mediated by endogenous NO and PGs, as well as capsaicin-sensitive sensory neurons, and to play a pivotal role in maintaining mucosal integrity during acid secretion.

Animals↗
PubMed1997

Irritant action of monochloramine in rat stomachs: effects of zinc L-carnosine (polaprezinc).

1. Effects of a novel zinc compound (polaprezinc), N-(3-aminopropionyl)-L-histidinato zinc, on the mucosal ulcerogenic response induced by ammonia (NH4OH) and monochloramine (NH2Cl) were examined in rat stomachs. 2. Oral administration (1 ml) of NH4OH (> 600 mM) and NH2Cl (> 60 mM) produced severe hemorrhagic lesions in unanesthetized rat stomachs, whereas hypochlorous acid (HClO) even at 120 mM did not cause any macroscopic damage. 3. Pretreatment of the animals with polaprezinc (2-12 mg/ml, 1 ml, PO) showed a dose-dependent inhibition against gastric lesions induced by NH4OH (1,800 nM) or NH2Cl (120mM), and this effect was significant at > 6 mg/ml in either case. These lesions were also significantly prevented by prior administration of dmPGE2 (2 micrograms/ml, 1 ml, PO). 4. Mucosal application of NH4OH (300 mM) and NH2Cl (10 mM) caused a marked reduction of transmucosal potential difference (PD) in ex vivo stomachs of anesthetized rats. The reduced PD responses caused by NH4OH and NH2Cl were prevented dose dependently by preexposure of the mucosa to polaprezinc, but not affected by dmPGE2. 5. Mucosal exposure to NH4OH (60 mM) caused a marked PD reduction in ex vivo stomachs made ischemic by bleeding from the carotid artery (1 ml per 100 g body wt), followed by severe gastric lesions. These ulcerogenic and PD responses caused by NH4OH plus ischemia were attenuated by prior application of polaprezinc as well as taurine (25 mg/ml, 1 ml), while dmPGE2 prevented the lesions without affecting the reduced PD response. 6. These results suggest that (a) NH2Cl damages the gastric mucosa at much lower concentrations than NH4OH, (b) polaprezinc protects the stomach against injury caused by either NH2Cl or NH4OH, and (c) the mechanisms underlying the protective action of polaprezinc remain unclear but may be different from those of dmPGE2.

Ammonium Chloride↗
PubMed1997

Expression of muscarinic receptor subtypes in rat gastric smooth muscle: effect of M3 selective antagonist on gastric motility and emptying.

Expression of muscarinic receptor subtypes in rat gastric smooth muscle was examined with reverse transcriptase-polymerase chain reaction (RT-PCR). Under the condition for detecting the messages of m1-m4 subtypes in brain, atrium, and gastric mucosa, only the fragments of m2 and m3 subtypes were amplified with RNA prepared from rat gastric smooth muscles. Furthermore, the amplified fragments were digested by restriction enzymes, reconfirming that the predicted size products of m2 and m3 contain the partial DNA sequences of m2 and m3 subtypes, respectively. We measured gastric motility in rats with a pressure transducer system under the continuous venous infusion of the muscarinic antagonists atropine and butylscopolamine (nonselective), AF-DX 116 (M2), zamifenacine (M3), and glucagon. Heart rate was monitored simultaneously in the tail. Gastric motility was inhibited in the presence of glucagon and zamifenacine without alteration of heart rate, whereas there was no inhibition in the presence of AF-DX 116 even after the augmentation of heart rate was observed. Gastric emptying was also suppressed in the presence of zamifenacine, which had an effect comparable with that of atropine, butylscopolamine, and glucagon. These results indicate that the activation of the M3 subtype in gastric smooth muscle causes its contraction, and the M3 selective antagonist could be a potentially useful drug without an adverse effect on the heart for radiological and endoscopic examination in the upper gastrointestinal tract.

Animals↗
PubMed1997

Mucosal ulcerogenic action of monochloramine in rat stomachs: effects of polaprezinc and sucralfate.

Effects of a novel zinc compound polaprezinc [N-(3-aminopropionyl)-L-histidinatozinc] and sucralfate on the mucosal ulcerogenic responses induced by monochloramine (NH2Cl) were examined in rat stomachs. Oral administration of NH2Cl (>60 mM) produced severe lesions in unanesthetized rat stomachs, with concomitant increase of lipid peroxidation. These lesions were aggravated by sensory deafferentation but not affected by pretreatment with indomethacin or L-NAME. The mucosal ulcerogenic response to NH2Cl was significantly inhibited by oral pretreatment with either dmPGE2 (10 microg/kg), capsaicin (30 mg/kg), or NOR-3 (3 mg/kg), the NO donor. Gastric lesions induced by NH2Cl were also inhibited by prior oral administration of polaprezinc (3-30 mg/kg) as well as sucralfate (30 and 100 mg/kg). The protective effect of polaprezinc was not affected by any pretreatments such as indomethacin, L-NAME, or sensory deafferentation, while that of sucralfate was significantly mitigated in the presence of either indomethacin or L-NAME. On the other hand, mucosal exposure to NH4OH (60 mM) caused a marked PD reduction in ex vivo stomachs made ischemic by bleeding from the carotid artery, followed by severe gastric lesions. These ulcerogenic and PD responses caused by NH4OH plus ischemia were also attenuated by prior application of polaprezinc, while dmPGE2 and sucralfate prevented such lesions without affecting the reduced PD response. These results suggest that: (1) NH2Cl generated either exogenously or endogenously damages the gastric mucosa, (2) both polaprezinc and sucralfate protect the stomach against injury caused by NH2Cl, and (3) the mechanisms underlying the protective action of sucralfate may be partly mediated by both endogenous PGs and NO but may be different from those of polaprezinc.

16,16-Dimethylprostaglandin E2↗
PubMed1997

Mechanism of acid secretory changes in rat stomach after damage by taurocholate: role of nitric oxide, histamine, and sensory neurons.

The present study was performed to investigate the mechanism underlying the acid stimulatory response in the stomach after damage under the inhibition of nitric oxide (NO) production by N(G)-nitro-L-arginine methyl ester (L-NAME). A rat stomach was mounted in an ex vivo chamber, perfused with saline, and the potential difference (PD) and acid secretion were measured before and after the application of 20 mM taurocholate (TC) for 30 min. Exposure of the stomach to TC caused a PD reduction and a decrease of acid secretion. Pretreatment with L-NAME did not affect basal acid secretion but significantly enhanced the acid secretion in the stomach after damage with TC, without any effect on the PD response. This effect of L-NAME was antagonized by simultaneous administration of L-arginine but not D-arginine. The luminal appearance of NO was significantly increased in the stomach after exposure to TC, and this change was completely blocked in the presence of L-NAME or when EGTA was applied together with TC. The enhanced acid secretory response to TC in the presence of L-NAME was inhibited by pretreatment with cimetidine, FPL-52694 (a mast cell stabilizer), or spantide (a substance P antagonist) or by chemical ablation of capsaicin-sensitive sensory neurons. Mucosal exposure to TC increased histamine output in the lumen and decreased the number of metachromatically staining cells in the stomach, and these changes were also significantly prevented by FPL-52694, spantide, or sensory deafferentation. These results suggest that 1) damage in the stomach may activate the acid stimulatory pathway in addition to the NO-dependent inhibitory mechanism, but the latter effect overcomes the former, resulting in a decrease in acid secretion, 2) the acid stimulation in the damaged stomach may be mediated by histamine released from the mucosal mast cell which may interact with capsaicin-sensitive sensory nerves, and 3) L-NAME unmasks the acid stimulatory response by suppressing the inhibitory mechanism.

Animals↗
PubMed1997

Gastric motility and mucosal ulcerogenic responses induced by prokinetic drugs in rats under prostaglandin-deficient conditions.

The present study was performed to examine whether gastric prokinetic drugs may induce damage in the rat stomach under normal and prostaglandin (PG)-deficient conditions. Male SD rats fasted for 18 hr were administered subcutaneously with three different prokinetic drugs such as metoclopramide (3-60 mg/kg), ondansetron (0.3-3 mg/kg), and cisapride (3-30 mg/kg). Half the number of these animals were pretreated with indomethacin (5 mg/kg) subcutaneously for induction of PG deficiency in the stomach. Administration of these drugs increased gastric motor activity in a dose-dependent manner and expedited gastric emptying at lower doses than those affecting gastric motility; the potency of the hypermotility effect was in the following order: metoclopramide = ondansetron > cisapride. None of these drugs alone caused gross damage in the stomach, although whitish rough areas were observed in the gastric mucosa along the folds. In the rats pretreated with indomethacin, however, both metoclopramide and ondansetron provoked multiple hemorrhagic lesions in the gastric mucosa. Indomethacin at this dose showed over 90% inhibition of cyclooxygenase activity without causing any damage in the stomach, and this PG-deficient effect was not affected by coadministration with the prokinetic drugs. The mucosal ulcerogenic responses induced by metoclopramide in the presence of indomethacin were significantly inhibited by prior administration of atropine (1 mg/kg) or PGE2 (300 micrograms/kg) at doses that inhibited gastric hypermotility induced by metoclopramide. These results suggest that: (1) gastric prokinetic drugs induce damage in rat stomachs under PG-deficient conditions at the doses that enhance gastric motility and emptying but not at the doses that expedite gastric emptying only, and (2) gastric hypermotility has the potential to cause gross damage in the stomach, supporting the importance of gastric motility as a pathogenic element of gastric lesions.

Animals↗
PubMed1997

Cyclo-oxygenase isozymes in mucosal ulcergenic and functional responses following barrier disruption in rat stomachs.

1. We examined the effects of selective and nonselective cyclo-oxygenase (COX) inhibitors on various functional changes in the rat stomach induced by topical application of taurocholate (TC) and investigated the preferential role of COX isozymes in these responses. 2. Rat stomachs mounted in ex vivo chambers were perfused with 50 mM HCl and transmucosal potential difference (p.d.), mucosal blood flow (GMBF), luminal acid loss and luminal levels of prostaglandin E2 (PGE2) were measured before, during and after exposure to 20 mM TC. 3. Mucosal application of TC in control rats caused a reduction in p.d., followed by an increase of luminal acid loss and GMBF, and produced only minimal damage in the mucosa 2 h later. Pretreatment with indomethacin (10 mg kg[-1], s.c.), a nonselective COX-1 and COX-2 inhibitor, attenuated the gastric hyperaemic response caused by TC without affecting p.d. and acid loss, resulting in haemorrhagic lesions in the mucosa. In contrast, selective COX-2 inhibitors, such as NS-398 and nimesulide (10 mg kg[-1], s.c.), had no effect on any of the responses induced by TC and did not cause gross damage in the mucosa. 4. Luminal PGE2 levels were markedly increased during and after exposure to TC and this response was significantly inhibited by indomethacin but not by either NS-398 or nimesulide. The expression of COX-1-mRNA was consistently detected in the gastric mucosa before and after TC treatment, while a faint expression of COX-2-mRNA was detected only 2 h after TC treatment. 5. Both NS-398 and nimesulide significantly suppressed carrageenan-induced rat paw oedema, similar to indomethacin. 6. These results confirmed a mediator role for prostaglandins in the gastric hyperaemic response following TC-induced barrier disruption, and suggest that COX-1 but not COX-2 is a key enzyme in maintaining 'housekeeping' functions in the gastric mucosa under both normal and adverse conditions.

Animals↗
PubMed1997

Megakaryocytes derived from CD34-positive cord blood cells produce interleukin-8.

In a serum-free liquid culture, thrombopoietin (TPO) selectively stimulated the growth of megakaryocytic cells from CD34-positive cord blood cells. Using these cultured cells, we investigated cytokine production by human megakaryocytes. Day 10 megakaryocytes (2 x 10(5)) secreted > 1000 pg/ml of interleukin (IL)-8, in contrast to small amounts of IL-1beta and IL-6. A time-course study showed that the IL-8 production of megakaryocytes occurred at the late phase of the culture period. The megakaryocyte-conditioned medium had the chemotactic potential of polymorphonuclear leucocytes, which was abrogated by the addition of anti-IL-8 antibody, suggesting the secretion of biologically active IL-8. The combination of TPO and IL-1alpha was required for a significant augmentation of the IL-8 secretion. Direct evidence for IL-8 synthesis in megakaryocytes was provided by reverse transcription-polymerase chain reaction on purified CD41b+ cells and by the detection of intracellular IL-8 in CD41b+ cells. These results suggest that TPO stimulates not only the proliferation and differentiation of the progenitors capable of megakaryocytic lineage expression but also IL-8 release by the megakaryocytic cells with the aid of IL-1.

Cells, Cultured↗
PubMed1997

Alterations in duodenal bicarbonate secretion and mucosal susceptibility to acid in diabetic rats.

BACKGROUND & AIMS: The gastroduodenal mucosal susceptibility to ulcerogenic stimuli increases in diabetic conditions, but the mechanism is unknown. The aim of this study was to investigate alterations in duodenal HCO3- secretory response in diabetic animals. METHODS: The experiments were performed in rats treated with streptozotocin (70 mg/kg intraperitoneally) after 1-6 weeks of diabetes, when blood glucose levels were > 300 mg/dL. HCO3 secretion was measured in the proximal duodenal loop of rats that were under urethane anesthesia using a pH-stat method. RESULTS: The duodenal HCO3 secretion induced by mucosal acidification was decreased in streptozotocin-treated rats depending on the duration of diabetes. The HCO3 secretion was also decreased in response to 16,16-dimethyl prostaglandin E2, vasoactive intestinal polypeptide, or vagal electrical stimulation. Acid load in the duodenum produced extensive damage in 5-6-week diabetic rats, although the same treatment caused only slight damage in the normal rat duodenum. Such alterations in the duodenal HCO3 secretory and ulcerogenic responses in diabetic rats were partially restored by daily injection of insulin. CONCLUSIONS: The results suggest that streptozotocin-diabetic conditions impair the duodenal HCO3- secretion in rats, possibly as a result of decreased sensitivity of the epithelial cell and dysfunction of neuronal pathway, thereby increasing the mucosal susceptibility to acid injury in the duodenum.

Animals↗
PubMed1997

Roles of prostaglandin E-receptor subtypes in gastric and duodenal bicarbonate secretion in rats.

BACKGROUND & AIMS: Receptors activated by prostaglandin (PG) E2 are pharmacologically subdivided into four subtypes (EP1-EP4). The EP-receptor subtype(s) involved in stimulation of gastroduodenal HCO3- secretion in rats were investigated. METHODS: Under urethane anesthesia, a stomach mounted in an ex vivo chamber or a proximal duodenal loop was perfused with saline, and HCO3- secretion was measured using a pH-stat method. RESULTS: Intravenous PGE2 increased HCO3- secretion by the gastroduodenal mucosa; this action was verapamil sensitive and, only in the duodenum, potentiated by isobutylmethyl xanthine (IBMX). Duodenal HCO3- secretion was stimulated by enprostil, sulprostone (EP1/EP3 agonist), misoprostol (EP2/EP3 agonist), and ONO-NT012 (EP3 agonist) but was not affected by butaprost (EP2 agonist) or 17-phenyl-PGE2 (EP1 agonist). Gastric HCO3- secretion was stimulated by sulprostone, enprostil, and 17-phenyl-PGE2 but not by misoprostol, butaprost, or ONO-NT012. SC-51089 (EP1 antagonist) inhibited the HCO3--stimulatory action of sulprostone only in the stomach. IBMX potentiated the HCO3- response to sulprostone in the duodenum, whereas verapamil reduced the response in both the stomach and duodenum. CONCLUSIONS: PGE stimulates HCO3- secretion via different EP-receptor subtypes in the stomach and duodenum: in the stomach, EP1 receptors are linked to Ca2+; in the duodenum, EP3 receptors are coupled with both adenosine 3', 5'-cyclic monophosphate and Ca2+.

1-Methyl-3-isobutylxanthine↗
PubMed1997

Localization of the phosphatidylserine-binding site of glyceraldehyde-3-phosphate dehydrogenase responsible for membrane fusion.

In this study, we demonstrated that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a phosphatidylserine (PS)-binding protein and localized the putative PS-binding site involved in the membrane fusion induced by this enzyme. In an attempt to identify the PS-binding proteins, we raised polyclonal antibodies against a 15-amino-acid synthetic peptide (amino acid residues 390-403 of phosphatidylserine decarboxylase), which was shown to bind specifically to PS. One polyclonal antibody, designated aPSD-2, crossreacted with GAPDH, and its binding to GAPDH was inhibited by PS but not by other phospholipids such as phosphatidylethanolamine and phosphatidylinositol. Kinetic analysis of GAPDH binding to phospholipid membranes by measuring surface plasmon resonance showed that GAPDH associated with the phospholipid membrane containing PS rapidly (k[on] =2.8 X 10(4) M(-1) X s[-1]) and dissociated extremely slowly (k[off]=5.9 X 10(-5) s[-1]), giving a low dissociation constant (KD=2.6nM). GAPDH bound less effectively to membranes without PS with a dissociation constants of 0.2 microM. GAPDH-induced vesicle fusion was also inhibited by aPSD-2, suggesting that this antibody recognizes the putative PS-binding site on GAPDH involved in the enzyme-induced membrane fusion. Chemical fragmentation of GAPDH with cyanogen bromide followed by separation and sequence analysis of the reactive peptide resulted in the identification of a single reactive peptide with the sequence of amino acid residues 45-103 of GAPDH. Analysis of aPSD-2 binding to synthetic peptides derived from the corresponding region localized the antibody-binding site to amino acid residues 70-94 of GAPDH. Both the 25-amino-acid synthetic peptide (amino acid residues 70-94 of GAPDH) and polyclonal antibody raised against this peptide inhibited GAPDH-induced membrane fusion, suggesting that these amino acid residues play a crucial role in this membrane fusion process.

Amino Acid Sequence↗
PubMed1997

Nitric oxide, histamine, and sensory nerves in the acid secretory response in rat stomach after damage.

The stomach normally responds to mucosa-damaging agents by decreasing acid secretion, but this acid response turn from "inhibition" into "stimulation" when the production of nitric oxide (NO) is inhibited by NG-nitro-L-arginine methyl ester (L-NAME). We investigated the mechanism underlying stimulation of acid secretion in the stomach after damage with taurocholate (TC) in the presence of L-NAME. A rat stomach was mounted in an ex vivo chamber and perfused with saline, and the potential difference (PD), luminal pH, and acid secretion were measured before and after application of 20 mM TC for 30 min. Exposure of the stomach to TC caused a reduction in PD, an increase in luminal pH, and a decrease in acid secretion. Pretreatment with L-NAME did not affect basal acid secretion but significantly increased secretion after damage with TC, without any effect on PD. This effect of L-NAME was antagonized by co-administration of L-arginine but not D-arginine. The luminal appearance of NO was also increased after exposure of the stomach to TC, a phenomenon completely blocked by L-NAME, or when EGTA was applied together with TC. The enhanced acid secretory response in the presence of L-NAME was inhibited by prior administration of cimetidine, FPL-52694 (a mast cell stabilizer), spantide (a substance P antagonist), or by chemical ablation of capsaicin-sensitive sensory neurons. Mucosal exposure to TC increased histamine output in the lumen and decreased the number of mucosal mast cells in the stomach. These changes were prevented by FPL-52694 or sensory neuronal ablation. These results suggest that (a) damage in the stomach may activate acid stimulation in addition to an NO-dependent inhibitory mechanism but that the latter effect overcomes the former, resulting in a decrease in acid secretion, (b) acid stimulation in the damaged stomach may be mediated by histamine released from the mucosal mast cells, a process that may interact with capsaicin-sensitive sensory nerves, and (c) L-NAME unmasks the acid stimulatory response by suppressing the inhibitory mechanism.

Animals↗
PubMed1997

Mechanism of gastric hyperemic response during acid secretion in rats: relation to nitric oxide, prostaglandins, and sensory neurons.

The mechanism of gastric mucosal hyperemic response during pentagastrin-induced acid secretion was investigated in anesthetized rats in relation to prostaglandins (PGs), nitric oxide (NO), and sensory neurons. A rat stomach was mounted in an ex vivo chamber and perfused with saline or glycine (200 mM), and the mucosal blood flow (GMBF), determined by laser Doppler flowmetry, and acid secretion was measured simultaneously. Intravenous infusion with a submaximal dose of pentagastrin (60 micrograms/kg/h) caused a significant increase in GMBF as well as acid secretion. Such GMBF responses were totally attenuated when acid secretion was inhibited by omeprazole and cimetidine or when the luminal H+ was buffered by mucosal perfusion with glycine (200 mM). Tripelennamine, an H1 antagonist, did not have any affect on acid secretory and GMBF responses to pentagastrin. On the other hand, prior administration of NG-nitro-L-arginine methyl ester (L-NAME), the NO synthase inhibitor, significantly mitigated the increase of GMBF induced by pentagastrin without any influence on acid secretion, and this effect was antagonized by simultaneous administration of L-arginine. The gastric hyperemic response to pentagastrin was also significantly mitigated by indomethacin or sensory deafferentation after capsaicin pretreatment, with no effect on acid secretion, and was totally inhibited by combined treatments with indomethacin plus L-NAME in addition to sensory deafferentation. Pentagastrin infusion for 8 h did not by itself cause macroscopic damage in the stomach, but additional treatments with L-NAME and indomethacin plus sensory deafferentation provoked severe lesions in the gastric mucosa. These results suggest that the gastric hyperemic response to pentagastrin (submaximal dose) is totally dependent on H+ and that this process is mediated by endogenous NO and PGs as well as by capsaicin-sensitive sensory neurons and plays a pivotal role in maintaining mucosal integrity during acid secretion.

Animals↗
PubMed1997

Monoclonal antibody #5-2-26 recognizes the phosphatase-sensitive epitope of rabies virus nucleoprotein.

We prepared monoclonal antibodies (MAbs) against the rabies virus N protein, among which one antibody (MAb 5-2-26) was shown to lack reactivity with the phosphatase-treated N protein. The MAb was able to recognize the sodium dodecyl sulfate (SDS)-denatured N protein. The MAb did not recognize the N-protein analogues produced in Escherichia coli (E. coli), indicating that the N-gene products were not normally processed in E. coli after translation. On the other hand, the MAb reacted normally with N-gene products produced in COS-7 cells, but not with those produced in the presence of K-252a (a protein kinase inhibitor of a broad spectrum). The MAb displayed weak cross-reactivity with the Triton-insoluble network structures composed of several components, while another phosphoprotein (M1) of the virus was not recognized at all. These results suggest that MAb 5-2-26 preferentially recognizes a phosphatase-sensitive linear epitope of N protein, which may enable further investigations to be conducted on the mechanism of N-protein phosphorylation and its role(s) in virus replication.

Animals↗
PubMed1997

Identification of a phosphatase-sensitive epitope of rabies virus nucleoprotein which is recognized by a monoclonal antibody 5-2-26.

We have investigated a phosphatase-sensitive sequential epitope of the nucleoprotein (N), one of the phosphoproteins of rabies virus, which is recognized by the monoclonal antibody (MAb) #5-2-26. The epitope was shared in common by all of the rabies virus strains we tested, including the HEP, ERA, CVS and Japanese strains (Nishigahara and Komatsukawa). Thin layer chromatography of the acid hydrolyzates of 32P-labeled N protein showed that the protein contained phosphoserine and phosphothreonine at a molar ratio of about 4 to 1, while no phosphotyrosine was detected. Immunoprecipitation studies with several deletion mutants of the N protein showed that the epitope is located in a region spanning from amino acid 344 to 415. If the phosphatase-sensitive epitope is located at or near the phosphoamino acid, the location of the latter could be narrowed further to a region from amino acid 354 to 389 by comparing the amino-acid sequences among the viral strains. To examine this assumption, point mutation was introduced by amino-acid substitution with alanine at either of five potential phosphorylation sites (i.e., positions 354, 375, 377, 386 and 389) in the 354-389 region. Among those, only one substitution, at position 389, greatly affected the antigenicity. Substitution of serine-389 by threonine also reduced the antigenicity. These results strongly suggest that serine-389 is a phosphorylation site and essential for constructing or stabilizing the antigenic structure for MAb 5-2-26.

Amino Acid Sequence↗