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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Departments of Physiology (T.B., Z.S., M.P., A.P.-B., D.D., S.K., R.P., S.J.K., W.W.P.) and Experimental Pharmacology (S.C.), Jagiellonian University Medical College, Cracow, Poland; Department of Biochemistry, Medical Academy, Gdansk, Poland (E.S.); and Department of Medicine I, University of Erlangen-Nuremberg, Erlangen, Germany (P.C.K.)
Received January 16, 2008; accepted April 1, 2008.
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), and an overexpression of mRNAs for cyclooxygenase (COX)-2 and CGRP in the gastric mucosa. R-3-(4-Fluoro-phenyl)-2-[5-(4-fluoro-phenyl)-benzofuran-2-ylmethoxycarbonylamino]-propionic acid (RO 324479), which is the selective antagonist of IP/PGI2 receptors, reversed the effects of MNA on gastric lesions and GBF. MNA-induced gastroprotection was attenuated by suppression of COX-1 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole; SC-560] and COX-2 [4-(4-methylsulfonylphenyl)-3-phenyl-5H-furan-2-one; rofecoxib] activity, capsaicin denervation, and by the pretreatment with CGRP8-37 or capsazepine. Addition of exogenous PGI2 or CGRP restored the MNA-induced gastroprotection in rats treated with COX-1 and COX-2 inhibitors or in those with capsaicin denervation. WRS enhanced MDA content while decreasing superoxide dismutase (SOD) activity in the gastric mucosa, but pretreatment with MNA reversed these changes. MNA exerts potent gastroprotection against WRS damage via mechanisms involving cooperative action of PGI2 and CGRP in preservation of microvascular flow, antioxidizing enzyme SOD activity, and reduction in lipid peroxidation.
; Maiese and Chong, 2003). Indeed, NA has been reported to exert several physiological and pharmacological effects, including the prevention of ATP depletion, inhibition of poly(ADP-ribose) polymerase, prevention of apoptosis, and suppression of lipid peroxidation (Klaidman et al., 1996, 2003). NA also possesses anti-inflammatory properties (Ogata et al., 2002; Ungerstedt et al., 2003) and exhibits angiogenic activity by promoting the growth of capillaries (Morris et al., 1989). 1-Methylnicotinamide (MNA) is the major primary metabolite of NA, which is formed in the liver by the enzyme nicotinamide N-methyltransferase, and it is further metabolized to 1-methyl-2-pyridone-5-carboxamide (M2PY) or 1-methyl-4-pyridone-5-carboxamide (M4PY) by aldehyde oxidase (Aoyama et al., 2000). For many years, MNA has been considered inactive. Recent studies, however, have indicated that MNA applied topically exhibits remarkable anti-inflammatory properties against skin diseases such as acne vulgaris, contact dermatitis (Gebicki et al., 2003), and rosacea (Wozniacka et al., 2005). Recently, it has been reported that MNA displays a potent antithrombotic activity in vivo that was mediated by the prostacyclin (PGI2) pathway (Chlopicki et al., 2007). By using the mouse model of contact hypersensitivity, it was demonstrated that MNA affords anti-inflammatory action that also seems to be mediated by PGI2 (Bryniarski et al., 2008). Little is known about the action of MNA in the gut; however, the gastric uptake of nicotinic acid via mechanism involving bilitranslocase was recently demonstrated, suggesting that significant amounts of niacin-related compounds could be taken up at the gastric level from food during digestion (Passamonti et al., 2000). Conversely, the gastroprotective effect of PGI2 and PGE2 is well established (Konturek et al., 1981). Accordingly, the recently discovered vasoprotective action of MNA mediated by PGI2 implies that there is a possibility for the involvement of MNA in gastric pathophysiology, particularly in the mechanism of gastric secretory functions, mucosal defense, and gastroprotection against acute gastric lesions.
The influence of MNA to the mechanism of gastric mucosal defense against the damage induced by noxious agents has not been studied so far. This prompted us to examine whether exogenous MNA affects the formation of stress-induced gastric damage, which is a serious clinical entity in humans ultimately resulting in gastric mucosal bleeding and erosions. Furthermore, the gastroprotective effect of MNA was compared with that exhibited by potent gastric acid inhibitors such as proton pump inhibitor omeprazole and histamine H2-receptor antagonist ranitidine against the formation of acute gastric lesions induced by stress. In a separate group of rats, the protective effects of MNA against the formation of ethanol lesions were determined to check whether MNA provided protection against acid-independent necrotizing type of gastric injury. An attempt was made to assess the mechanism of gastroprotective action of MNA by focusing on the involvement of PGI2 and sensory nerves. We measured the gastric mucosal generation of PGI2 (6-keto-PGF1) and by assessment of mRNA expression for COX-1 and COX-2 as well as by pharmacological tools such as specific IP/PGI2 receptor antagonist RO 324479 (Bley et al., 2006), the nonselective (indomethacin) and selective inhibitors of COX-1 (SC-560) and COX-2 [4-(4-methylsulfonylphenyl)-3-phenyl-5H-furan-2-one rofecoxib] activity. In addition, an attempt was made to determine the involvement of sensory nerves in MNA-induced gastroprotection, considering that reciprocal interactions between activities of PGI2 and sensory nerve-derived CGRP have been reported previously (Holzer et al., 1991; Arai et al., 2003). The mechanism of gastroprotection induced by MNA in water restraint stress (WRS) model of gastric damage was determined by studying the influence of MNA on the gastric blood flow (GBF), plasma levels of MNA assessed by liquid chromatography-mass spectrometry, antioxidizing enzyme superoxide dismutase (SOD), and malonylodialdehyde (MDA) content in the gastric mucosa, as an index of lipid peroxidation.
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Gastric Secretory Studies. The effects of MNA (synthesized in the Institute of Applied Radiation Chemistry Technical University of Lodz, Lodz, Poland) on gastric acid secretion were examined in 40 conscious rats equipped approximately 1 month earlier with a Thomas-type gastric fistula (GF) as described previously (Brzozowski et al., 2000). The animals were fasted overnight, but they had free access to water 24 h before the experiment. They were placed in individual Bollman-type cages to maintain the minimum restraint necessary. The gastric fistulas were opened and the stomachs were gently rinsed with approximately 5 ml of tap water at 37°C. The basal gastric secretions were then collected for 60 min, and MNA and NA were administered intragastrically (i.g.) in one of doses ranging from 6.25 to 100 mg/kg, with each dose being dissolved in 1 ml of saline and administered on a separate test day. In control tests, vehicle (1 ml of saline i.g.) was given in the same dose as in tests with MNA or NA, with the collection of gastric juice being continued for the final 60 min. Vehicle (1 ml of saline) was applied i.g. in control animals parallel to animals administered with MNA and NA. The volume and acid concentration of each collected sample of gastric juice were measured, and acid outputs (expressed in term of micromoles of acid per 30 min) and pepsin output (expressed in term of milligrams per 30 min) were determined as described previously (Brzozowski et al., 2000).
Gastroprotection Studies and Measurement of GBF. Acute gastric lesions were induced by exposing animals to 3.5 h of water immersion and WRS according to the procedure described by our group previously (Brzozowski et al., 2004). In addition, the efficacy of MNA applied i.g. to prevent gastric injury induced by corrosive agent such as ethanol was examined in an acute model of gastric damage induced by i.g. application of 75% ethanol in a volume of 1 ml using a metal catheter (Brzozowski et al., 2006). At 1 h upon the ethanol application, or 3.5 h upon the termination of WRS, the animals were lightly anesthetized with phenobarbital (60 mg/kg i.p.), their abdomens were opened by a midline incision, and the stomachs were exposed for the purpose of measuring GBF by means of H2 gas clearance technique as described previously (Brzozowski et al., 1999). For this purpose, double electrodes of electrolytic regional blood flowmeter (model RBF-2; Biotechnical Science, Osaka, Japan) were inserted into the gastric mucosa. The measurements were made in three areas of the oxyntic mucosa, and the mean values of the measurements were calculated and expressed as percentage of changes of those recorded in the vehicle (saline)-treated animals. After the GBF measurement, the venous blood samples were withdrawn from the vena cava for the determination of plasma levels of MNA as described below, and the stomach was removed, rinsed with saline, and pinned open for macroscopic examination. Rats were sacrificed at 1 h after i.g. ethanol instillation and 3.5 h after the end of WRS. Their stomachs were excised and opened along the greater curvature, and the number of gastric bleeding erosions in case of WRS lesions and the area of ethanol-induced gastric lesions expressed in square millimeters were determined by computerized planimetry (Morphomat; Carl Zeiss, Jena, Germany) (Brzozowski et al., 1996) by an individual who was unaware of the rat groupings.
In some experiments with i.g. administration of MNA with or without 75% ethanol application or exposure to 3.5 h of WRS, the standardized specimens from the corpus of the stomachs were fixed in 10% buffered formalin, and the paraffin sections were stained with hematoxylin-eosin for histology evaluation (Brzozowski et al., 2006). A Nikon microscope equipped with Microplan II digital image system was used for the quantitative histology examination (morphometry) of the sections. Coded specimens of mucosa stained with hematoxylin and eosin were evaluated quantitatively at 500x magnification under blinded conditions.
Involvement of COX-PGI2 and Sensory Nerve Systems in Gastroprotection Induced by MNA. In subsequent studies four major series (A, B, C, and D) of experiments were carried out. Series A was used to assess the effect of exogenous MNA given i.g. (6.25–100 mg/kg). For comparison purposes, NA was administered i.g. in single doses of 50 or 200 mg/kg against the mucosal lesions induced by WRS or ethanol, respectively. In series B and C, the involvement of COX-PGI2 and the efficacy of antisecretory agents, namely, omeprazole, a proton pump inhibitor, and ranitidine, a histamine H2-receptor antagonist, in protection afforded by MNA against WRS-induced mucosal damage was determined. To establish the role of PGI2 in the MNA-induced gastroprotection, we used a specific IP receptor antagonist, RO 324479, that has been described recently (Bley et al., 2006). RO 324479 was shown to be devoid of a significant effect on prostaglandin receptors and many other receptors, and its in vivo efficacy to blunt PGI2-mediated responses has been demonstrated previously (Bley et al., 2006; Bryniarski et al., 2008).
Several groups of rats, each consisting of six to eight animals, were pretreated 30 min before the WRS either with 1) vehicle (saline); 2) MNA (standard dose 50 mg/kg i.g.); 3) RO 324479 (10 mg/kg i.p.); 4) SC-560 (5 mg/kg i.g.), a selective COX-1 inhibitor (Brzozowski et al., 1999); 5) rofecoxib (10 mg/kg i.g.), a highly selective COX-2 inhibitor (Takeuchi et al., 2004); or 6) indomethacin (5 mg/kg i.p.), a nonselective COX inhibitor (Brzozowski et al., 1999). At the dose used in the present study, indomethacin has been shown in previous studies to inhibit gastric PGE2 generation by 
90% without itself causing any mucosal damage (Konturek et al., 1981; Brzozowski et al., 1999). The doses of SC-560 and rofecoxib were selected on the basis of previous studies showing that these agents almost completely suppressed PGE2 generation in exudates of air-pouch inflammation and inhibited gastric PGE2 production in mucosa with pre-existing gastric ulcer (Brzozowski et al., 2001; Takeuchi et al., 2004). SC-560 (Cayman Chemical, Ann Arbor, MI) or rofecoxib (Merck Sharp and Dhome, Warsaw, Poland) was first dissolved in absolute ethanol or methanol to obtain stock solutions of 50 mg/ml or 75 mg/ml, and then it was diluted to the desired concentrations with isotonic saline. Control rats received the corresponding vehicle. Our preliminary studies (data not included) showed that none of the COX inhibitors used in this study alone produced any gastric lesions at the doses tested. In addition, in separate groups of rats the effect of antisecretory agents omeprazole and ranitidine applied in a comparable dose of 20 mg/kg i.g. was compared with that of MNA applied in a standard dose of 50 mg/kg i.g. The dose of these antisecretory agents was selected on the basis of our previous study (Brzozowski et al., 2000) showing that omeprazole and ranitidine were highly effective in the attenuation of acute gastric mucosal lesions induced by ischemia-reperfusion.
Samples of the oxyntic gland area were taken by biopsy (approximately 100 mg) immediately after the animals were euthanized to determine the mucosal generation of 6-keto-PGF1, the stable metabolite of PGI2. After 30 min of incubation of the biopsies of gastric mucosa in Eppendorf tubes (37°C), samples of effluent were taken for analysis as described previously (Whittle et al., 1990). Samples were stored at –70°C until assayed by commercially available enzyme immunoassay kits (R&D Systems, Minneapolis, MN) (Chlopicki et al., 2007), and the concentration of 6-keto-PGF1 was expressed in picograms per milliliter per milligram of wet tissue weight.
The role of sensory afferent nerves (series D) and neuropeptides such as CGRP released from sensitive afferent nerve endings in gastroprotection by MNA was tested in rats with capsaicin-induced deactivation of these nerves (Konturek et al., 1995; Brzozowski et al., 1996) or in those pretreated either with capsazepine, the antagonist of transient receptor potential vanilloid type 1 (TRPV1) (Caterina et al., 1997), or the antagonist of CGRP receptors, CGRP8-37 (series C) (Konturek et al., 2000). Chemical ablation of sensory afferent nerves was achieved with capsaicin (Sigma-Aldrich, St. Louis, MO) injected s.c. for three consecutive days at a respective dose of 25, 50, and 50 mg/kg (total dose of 125 mg/kg) approximately 2 weeks before the experiment (Holzer et al., 1991; Konturek et al., 1995). Capsazepine was dissolved in 10% Tween 20 and 10% ethanol with normal saline. Both capsazepine and CGRP antagonist CGRP8-37 were applied 30 min before i.g. administration of MNA or vehicle followed 30 min later by WRS. The experimental protocol included the following study groups, each consisting of six to eight animals: 1) vehicle (saline 1 ml i.g.) followed 30 min later by WRS in rats with intact afferent nerves; 2) MNA (standard dose 50 mg/kg i.g.) followed 30 min later by WRS in rats with intact sensory nerves; 3) vehicle (saline 1 ml i.g.) followed 30 min later by WRS in rats with capsaicin deactivated afferent nerves or those pretreated with capsazepine (5 mg/kg i.g.) or CGRP8-37 (100 µg/kg i.p.); and 4) MNA (50 mg/kg i.g.) followed 30 min later by WRS in rats with capsaicin-deactivated afferent nerves or those pretreated with capsazepine (5 mg/kg i.g.) or CGRP8-37 (100 µg/kg i.p.).
Determination of Plasma MNA Levels in Rats Exposed to WRS with or without MNA Pretreatment. As mentioned, immediately after GBF measurement, a venous blood sample was withdrawn from the vena cava into EDTA-containing vials, and the concentration of endogenous MNA and its metabolites in plasma samples was measured using liquid chromatography mass spectrometry as described previously (Slominska et al., 2006). Chromatographic separation was performed using 3-µm Hypersil C18-BDS 150 x 2.0-mm column. Buffer A was 10 mM nonafluoropentanoic acid in water, and buffer B was 100% acetonitrile. The mobile phase was run at 0.2 ml/min in a gradient from 0 to 60% B in 12 min. The mass detector (LCQ Advantage; Thermo Electron Corporation, Waltham, MA), with an electrospray ion source, was operated in a positive single-ion monitoring mode for detection of [M + H]+ species of NA, MNA, M2PY, and M4PY, with the collision energy setting at 25%. Internal standard (2-chloroadenosine) signal was extracted from full MS mode. Electrospray cone voltage was set at 4.5 kV, and heated capillary temperature was 275°C. Sheath gas flow was set at 35 arbitrary units. The ion optics was optimized using standard instrument procedures during infusion of NA. Rat plasma was deproteinized using 10% trichloroacetic acid followed by ether extraction. Recovery of M2PY, M4PY, and NA added to the samples with known concentration was 75 to 95%. The coefficient of variation was below 10% for repeated injections on the same day. However, a much larger (>20%) variation was observed for repeated injections between days (Slominska et al., 2006).
Determination of Lipid Peroxidation and SOD Activity and in Gastric Mucosa Exposed to WRS with and without Pretreatment with MNA. Given that lipid peroxidation is a well established mechanism of cellular injury induced by reactive oxygen metabolites, we measured the changes in the MDA as an indicator of the lipid peroxidation in gastric mucosa exposed to WRS with and without treatment with MNA. For the measurement of lipid peroxidation, each gastric mucosa sample was weighed, transferred to the ice-cooled test tube, and homogenized in 400 µl of 20 mM Tris buffer, pH 7.4, containing 5 mM butylated hydroxytoluene to prevent new lipid peroxidation, which can occur during homogenization. The homogenate was then centrifuged at 4°C for 10 min, and resultant supernatant (200 µl) was stored at –80°C until an assay of lipid peroxidation. The content of lipid peroxidation was measured at 37°C by spectrophotometer at wavelength of 586 nm and compared with the absorbance of purified MDA as the standard (Kwiecie et al., 2004).
The activity of SOD was measured in the gastric mucosa of rats exposed to WRS with or without pretreatment with MNA using SOD 525 assay kits (OXIS Research, Inc., Portland, OR) (Konturek et al., 2000; Kwiecie et al., 2004). The gastric tissue was first washed with 0.9% NaCl containing 0.16 mg/ml heparin to remove red blood cells, which potentially could have interfered with the SOD activity in the gastric specimens. Then, the biopsy sample was blotted on the filter paper, weighed, and finally homogenized in 400 µl of phosphate-buffered saline buffer, pH 7.4, using a Tissuemizer Ultra Turax (Janke & Kunkel, Staufen, Germany). The principle of the SOD measurement is based on the SOD-mediated increase in the rate of autooxidation of 5,6,6a,11b-tetrahydro-3,9,10-trihydrobenzoflurene in aqueous solution at 37°C to yield a chromophore with a maximal absorbance at 525 nm. Ethanol-chloroform extraction was used to inactivate manganese-SOD and iron-SOD and to ensure that the assay is specific for copper/zinc-SOD.
Reverse Transcriptase-Polymerase Chain Reaction for Detection of mRNA for COX-1, COX-2, and CGRP in Rats Exposed to WRS with or without MNA Pretreatment. The stomachs were removed from intact rats and from those treated with vehicle (control) or MNA with or without exposition to WRS for the determination of COX-1, COX-2, and CGRP mRNA expression by RT-PCR using specific primers. Gastric mucosal specimens were scraped off from oxyntic mucosa with the use of a slide glass, and they were immediately snap-frozen in liquid nitrogen and stored at –80°C until analysis. Total RNA was extracted from mucosal samples by a guanidinium isothiocyanate/phenol chloroform method using kit from Stratagene (Heidelberg, Germany). Using 1% agarose-formaldehyde gel electrophoresis and ethidium bromide staining, the total RNA concentration in each sample was determined. Aliquoted RNA samples were stored at –80°C until analysis.
Single-stranded cDNA was generated from 5 µg of total cellular RNA using StrataScript reverse transcriptase and oligo(dT) primers (Stratagene). In brief, 5 µg of total RNA was uncoiled by heating (65°C for 5 min), and then it was reversed by transcribing into cDNA in a 50-µl reaction mixture that contained 50 U of Moloney murine leukemia virus reverse transcriptase; 0.3 mg of oligo(dT) primer; 1 ml of RNase block ribonuclease inhibitor (40 U/µl); 2 ml of a 100 mM mixture of deoxyadenosine triphosphate, deoxyribothymidine triphosphate, deoxyguanosine triphosphate, and deoxycytidine triphosphate; and 5 ml of 10x RT buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, and 5 mM MgCl2). The resultant cDNA (2 µl) was amplified in a 50-µl reaction volume containing 0.3 ml (2.5 U) of Taq polymerase, 200 mM (each) dNTP (GE Healthcare, Chalfont St. Giles, UK), 1.5 mM MgCl2, 5 ml of 10x polymerase chain reaction buffer (50 mM KCl and 10 mM Tris-HCl, pH 8.3), and primers used at final concentration of 0.5 mM. The mixture was overlaid with 25 µl of mineral oil to prevent evaporation. The polymerase chain reaction mixture was amplified in a DNA thermal cycler (PerkinElmer Life and Analytical Sciences, Waltham, MA) in the area dedicated for performing PCR reaction. The polymerase chain reaction mixture was amplified in a DNA thermal cycler (PerkinElmer Life and Analytical Sciences), and the incubation and thermal cycling conditions were as follows: denaturation at 94°C for 1 min, annealing at 60°C for 45 s, and extension at 72°C for 2 min. The number of cycles was 30 for β-actin, 32 for COX-1, 33 for COX-2, and 31 for CGRP. The nucleotide sequences of the primers for COX-1 and COX-2 were selected on the basis of the published cDNA encoding COX-1, COX-2, and β-actin (Brzozowski et al., 2001, 2006), respectively, and they were synthesized by Invitrogen (Eggenstein, Germany). The nucleotide sequences of the primers for CGRP were identical to those published by Peng et al. (2002). Polymerase chain reaction products were detected by electrophoresis on a 1.5% agarose gel containing ethidium bromide. Location of predicted products was confirmed with the use of DNA 100-base pair ladder (Invitrogen) as a standard size marker. The intensity of bands was quantified using densitometry (LKB Ultrascan; GE Healthcare) as described in details in a previous study (Konturek et al., 2000). The signals for COX-1 and COX-2 mRNAs were standardized against the β-actin signal for each sample, and results are expressed as COX-1 and COX-2/β-actin mRNA ratio.
Statistical Analysis. Results are expressed as means ± S.E.M. Statistical analysis was done using analysis of variance and the two-way analysis of variance test with a Tukey's post hoc test where appropriate. Differences of p < 0.05 were considered significant.
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Effects of Pretreatment with MNA on WRS- and Ethanol-Induced Lesions and the Alterations in the GBF and Plasma MNA. The results of i.g. administration of MNA on the mean number of WRS-induced gastric lesions as well as accompanying changes in the GBF and plasma MNA levels are presented in Fig. 1. Such pretreatment with MNA applied i.g. dose-dependently reduced the number of gastric lesions, which were evoked by 3.5 h of WRS, with the threshold reduction occurring at a dose of 12.5 mg/kg, and with the ID50 value averaging about 46 mg/kg MNA. The reduction of the lesion number of WRS damage by MNA was accompanied by a significant and dose-dependent rise in the GBF and plasma MNA levels (Fig. 1). Pretreatment with NA (50 mg/kg i.g.) also significantly reduced the number of WRS lesions, although this reduction in the number of WRS lesions was significantly less pronounced in comparison with that of MNA applied in the similar dose. The representative macroscopic example of the stomach pretreated with vehicle (saline) or MNA (50 mg/kg i.g.) is presented in Fig. 2. A dramatic reduction in the number of WRS-induced gastric lesions is observed in MNA-pretreated gastric mucosa compared with that pretreated with vehicle. As shown in Fig. 3, i.g. application of 75% ethanol resulted in the formation of gastric lesions followed by the significant reduction in GBF by approximately 30% compared with the intact gastric mucosa. With graded doses of MNA administered i.g. (12.5–200 mg/kg) before ethanol, the area of ethanol-induced gastric lesions was significantly attenuated, and a significant increase in the GBF starting with 25 mg/kg MNA was recorded (Fig. 3). As shown in Table 2, histologically, the exposure of gastric mucosa to WRS and ethanol in rats pretreated with vehicle resulted in a widespread denudation of mucosal surface and deep necrosis. However, rats pretreated with MNA applied i.g. in a standard dose of 50 mg/kg showed a significant reduction in the area of denuded surface and deep necrosis compared with those pretreated with vehicle. The administration of omeprazole significantly attenuated WRS-induced gastric lesions while raising the GBF; these effects were significantly more pronounced than those caused by MNA applied in a standard dose of 50 mg/kg (Fig. 4). In this dose used, MNA was almost equally effective in attenuation of WRS lesions, and it caused a similar increase in the GBF compared with those evoked by histamine H2-receptor antagonist ranitidine (Fig. 4).
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Effect of Selective IP Receptor Inhibition and Nonselective and Selective COX-1 and COX-2 Inhibitors on MNA-Induced Gastroprotection against WRS-Induced Gastric Damage and Alteration in GBF. As shown in Fig. 5, exposure to WRS resulted in a significant decrease in the mucosal generation of 6-keto-PGF1, compared with that measured in the intact gastric mucosa. This effect was partially reversed in rats pretreated with MNA (50 mg/kg i.g.). The increase in the mucosal generation of 6-keto-PGF1 was observed in MNA-treated gastric mucosa, which was completely suppressed in animals treated with indomethacin (5 mg/kg i.p.). The reduction in WRS-induced gastric damage and accompanying increase in the GBF evoked by MNA were abolished by the pretreatment with RO 3244794, the selective antagonist of IP receptors (Fig. 6). As shown in Fig. 7, MNA resulted in a similar attenuation in the number of gastric lesions induced by WRS and similar rise in GBF as that shown in Fig. 1. Indomethacin (5 mg/kg i.p.), which by itself significantly aggravated gastric lesions, induced WRS, and produced a significant fall in GBF, in comparison with vehicle-pretreated animals, abolished the reduction in the number of the lesions and the accompanying rise in GBF and mucosal PGI2 generation evoked by MNA (Figs. 5 and 7). The decrease in the number of WRS lesions and accompanying increase in GBF caused by MNA were also significantly attenuated by pretreatment with SC-560 (5 mg/kg i.g.) and rofecoxib (10 mg/kg i.g.), the selective COX-1 and COX-2 inhibitors, respectively. The concurrent treatment with exogenous PGI2 (10 µg/kg i.g.) restored the gastroprotective and hyperemic activity of MNA in stressed rats treated with COX-1 and COX-2 inhibitors that were exposed to WRS (Fig. 7).
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Effect of MNA on the Plasma CGRP Levels, Mucosal MDA Content, and SOD Activity in Rats Exposed to WRS. Table 3 indicates the changes in plasma levels of CGRP and in the mucosal content of MDA plus 4-HNE, measured as an index of lipid peroxidation, and in the mucosal activity of SOD in the gastric mucosa of WRS animals with or without pretreatment with vehicle or MNA. In nonstressed rats treated with MNA, the significant increase in the plasma CGRP levels compared with that in intact animals without any significant alteration in gastric mucosal MDA plus 4-HNE content was observed (Table 3). The exposure of rats to 3.5 h of WRS resulted in a significant decrease in the plasma levels of CGRP, a significant rise in the mucosal MDA plus 4-HNE content, and a significant fall in the mucosal SOD concentration compared with respective values measured in the intact gastric mucosa. In contrast, pretreatment with MNA applied i.g. in a standard dose of 50 mg/kg significantly enhanced plasma CGRP concentration, significantly attenuated the mucosal MDA plus 4-HNE content, and in part, restored the SOD activity compared with those values attained in vehicle-control animals exposed to WRS alone (Table 3).
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Effect of Capsaicin Denervation and Treatment with Capsazepine and CGRP8-37 on MNA Afforded Gastroprotection and Hyperemia against WRS-Induced Gastric Damage. Deafferentation with parenteral pretreatment with neurotoxic dose of capsaicin (approximately 2 weeks before the experiment) significantly increased the number of WRS-induced lesions, and it considerably reduced the GBF compared with the vehicle-treated rats with intact sensory nerves (Fig. 8). In rats with capsaicin deafferentation, the protective activity of MNA applied in a standard dose of 50 mg/kg i.g. and accompanying rise in the GBF and plasma levels of MNA were significantly reduced compared with those in rats with intact sensory nerves. Administration of CGRP alone in a dose of 10 µg/kg s.c. resulted in a small but significant reduction in the number of WRS-induced gastric lesions in rats with intact sensory nerves without capsaicin treatment (22 ± 2.6 in vehicle pretreated versus 16 ± 1.8 in CGRP pretreated). The concurrent administration of CGRP (10 µg/kg s.c.) with MNA in rats with capsaicin denervation restored the protection and accompanying rise in the GBF and plasma MNA levels to the extent similarly observed in MNA-treated rats with intact sensory nerves.
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It is interesting that MNA exhibited dose-dependent reduction in WRS- and ethanol-induced gastric lesions, and this gastroprotective activity was much more pronounced than that exerted by NA, a biological precursor of MNA. With respect to NA, this activity, in keeping with the previous observation that NA has been originally recognized as an important cofactor for the formation of dinucleotide, exhibits anti-inflammatory and radical scavenging activity (Ogata et al., 2002) and affords neuroprotection against hypoxic brain injury and thermal and spinal cord damage (Smith et al., 1989; Feng et al., 2006). Moreover, the activation of adenylyl cyclase/protein kinase A system facilitates a neural release of NA adenine dinucleotide in the mesenteric artery system, suggesting the role for derivatives of NA, in the control of the vascular circulation in the upper gastrointestinal tract (Bobalova and Mutafova-Yambolieva, 2006). It is of interest that the protective effects of MNA against WRS-induced gastric damage were accompanied by a significant and dose-dependent rise in the plasma MNA increments, and marked attenuation of the fall in the gastric blood flow provoked by WRS and ethanol, suggesting that MNA-evoked increase in the gastric microcirculation could be an important component of the protective action of this amide in the stomach. This is supported by our preliminary observation that a single application of MNA results in profound time-dependent increase in plasma MNA concentrations, reaching the peak at 3 h with only small increments in plasma concentrations of other MNA metabolites, such as Met4PY and Met2PY (data not shown). The activity of antioxidizing enzyme SOD, which was diminished in gastric mucosa of stressed animals, was preserved by treatment with MNA. Furthermore, the MDA content, which was significantly raised in the gastric mucosa of rats subjected to WRS, was significantly reduced in rats pretreated with MNA, suggesting that amelioration of WRS-induced gastric lesions by MNA depends upon the antioxidizing activity and attenuation of the lipid peroxidation process in gastric mucosa of stressed animals.
The results of secretory studies revealed that MNA applied in the doses that were gastroprotective against WRS injury dose-dependently inhibited gastric acid and pepsin secretion in well adapted conscious rats provided with GF, suggesting that its acid inhibitory effect could contribute to the gastroprotective effect of this amide. This is consistent with the previous report that NA, the precursor of MNA, inhibits gastric secretion after oral administration in humans (Stratford et al., 1996). In our study, omeprazole, the proton pump inhibitor, was superior to both MNA and the histamine H2-receptor antagonist ranitidine, but MNA exhibited comparable activity with ranitidine in attenuation of WRS-induced lesions. This antisecretory action could contribute to gastroprotection by MNA against acid-dependent WRS damage, but it cannot serve as satisfactory explanation for the efficacy of MNA to attenuate ethanol-induced gastric lesions, in which gastric acid plays a minor role. Further studies are necessary to explain the mechanism of gastroprotective action of this amide against necrotizing type of gastric lesions.
Arachidonate metabolites were thought to act as the classic mediators of cytoprotection (Robert, 1979) cooperating together with NO and sensory nerves in the mechanism of gastric mucosal defense (Whittle et al., 1990). Recent studies militate, however, against the role of NO in the action of MNA because the suppression of NO activity by N
-nitro-L-arginine methyl ester failed to modify the MNA-induced thrombolytic activity, but it involves PGI2 formation due to an activation of COX-2 activity (Chlopicki et al., 2007). This is why we tested whether MNA affects the generation of mucosal 6-keto-PGF1, a stable metabolite of PGI2 in the gastric mucosa, and we used rats pretreated with a selective antagonist of IP receptors, RO 3244794. First, we established that MNA-induced protection and hyperemia are accompanied by the enhancement in the mucosal generation of 6-keto-PGF1 in the presence of WRS, an effect consistent with the disappearance of MNA-induced protection and mucosal hyperemia in RO 3244794-pretreated animals. Moreover, MNA-induced gastroprotection was accompanied by an overexpression of mRNA for COX-2, whereas the expression of COX-1 mRNA remained unchanged. It is interesting that WRS decreased gastric mucosal PGI2 while increasing the expression of COX-2 mRNA, and this effect was further enhanced by MNA, possibly resulting from a mucosal compensatory effect of PGI2 depletion observed in stressed animals. The suppression of COX-1 and COX-2 activity by nonselective COX inhibitor indomethacin or SC-560 and rofecoxib (Brzozowski et al., 2001; Takeuchi et al., 2004), respectively, greatly attenuated the protective and hyperemic effects of MNA, indicating that endogenous PG derived from the COX-1 and COX-2 pathway contribute to the beneficial effects of this amide in the stomach exposed to WRS.
Capsaicin-sensitive afferent nerves releasing vasodilatory neuropeptides play a central role in the gastroprotection of the stomach (Stroff et al., 1995; Brzozowski et al., 1996). Activation of sensory nerves influences the secretory functions in the stomach and exhibits gastroprotection by increasing gastric mucosal blood flow via the release of CGRP from sensory afferent nerves (Holzer et al., 1991; Kato et al., 1996). Given that NA and its metabolites reverse the symptoms of pellagra (pellagra preventive vitamin PP also known as vitamin B3) and they were reported to exert cytoprotective effects in the neural, vascular, and dermal tissues, we attempted to test the hypothesis that MNA protection involves the activation of afferent sensory nerves leading to the release of CGRP. Indeed, one of such neurotransmitter could be CGRP, because in our study, the MNA-induced protection and accompanying hyperemia were attenuated by CGRP8-37, a CGRP receptor antagonist that was shown previously to inhibit protective effect of exogenous gastrin, leptin, and ghrelin as well as those released endogenously by peptone meal and cholecystokinin (Konturek et al., 1995; Brzozowski et al., 2004). The binding places for the capsaicin, a selective stimulator of these afferent neurons, has been identified and named TRPV1 (Caterina et al., 1997). The capsaicin activation of TRPV1 could be abolished by capsazepine (Harada and Okajima, 2007). It was proposed that CGRP released from sensory nerve endings increases the production of PGs, especially PGI2, thus reducing stress-induced gastric damage (Shimozawa et al., 2006; Harada and Okajima, 2007). Since the mechanism of gastric mucosal defense includes cooperation between PG and sensory neuropeptides (Whittle et al., 1990, Brzozowski et al., 1996), the MNA-induced gastroprotection involving PG may originate not only from direct action but also from the activation of afferent sensory neurons by this agent. Indeed, MNA enhanced local expression of mRNA for CGRP, and it triggered release of this neuropeptide into the circulation as reflected by the enhancement in the plasma CGRP levels in MNA-pretreated animals. Furthermore, the capsaicin-deactivation of primary afferent nerves eliminated the protective activity of MNA, significantly attenuated the plasma increments of MNA, and abolished the rise in GBF induced by this amide, and these effects were restored by CGRP administered in rats with capsaicin denervation. These findings indicate that sensory nerves are essential for microcirculatory response and of significant importance for the gastroprotective activity of MNA. The protective and hyperemic activity of MNA was mitigated by capsazepine, an antagonist of TRPV1, suggesting that MNA stimulates the afferent nerves through the activation of TRPV1 directly or indirectly, possibly via enhancement in PGI2 generation, resulting in the liberation of CGRP. MNA-induced gastroprotection against damage induced by WRS might be due to the recruitment of endogenous endothelial and/or gastric mucosal PGI2 that cooperates with CGRP released from afferent sensory nerves. Our study indicates that MNA might be of clinical interest and deserves further clinical study trial because in addition to the recognized thrombolytic activity, this agent affords gastroprotection against acute gastric lesions.
In summary, these results demonstrate that administration of exogenous MNA, which is accompanied by a significant plasma increment of this amide, exhibits dose-dependent gastroprotection against the WRS-induced lesions. Evidence was provided that these protective and hyperemic effects of MNA against stress injury may involve cooperation between sensory nerves possibly sensitized by endogenous PGI2 to release CGRP acting via activation of TRVP1 receptors. Because gastroprotection by MNA was accompanied by the rise in the plasma levels of this amide, it is suggested that MNA may act locally to activate the above-mentioned protective mechanisms and to strengthen the gastric mucosal defense in animals exposed to adverse conditions such as stress.
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The article contains the data from Kwiecién S, Brzozowski T, Konturek PC, Sliwowski Z, Chlopicki S, Slonimska E, Gebicki J, Konturek SJ, and Pawlick WW (2007) Role of prostaglandin (PG), nitric oxide (NO) and lipid peroxidation in the gastroprotective and ulcer healing activities of 1-methylnicotinamide (MNA). Gastroenterology 132 (Suppl. 2): A411–A412.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: NA, nicotinamide; MNA, 1-methylnicotinamide; M2PY, 1-methyl-2-pyridone-5-carboxamide; M4PY, 1-methyl-1–4-pyridone-5-carboximide; PGI2, prostacyclin; PGE2, prostaglandin E2; COX, cyclooxygenase; RO 3244794, R-3-(4-fluoro-phenyl)-2-[5-(4-fluoro-phenyl)-benzofuran-2-ylmethoxycarbonylamino]-propionic acid; CGRP, calcitonin gene-related peptide; WRS, water immersion and restraint stress; GBF, gastric blood flow; SOD, superoxide dismutase; MDA, malonyldialdehyde; GF, gastric fistula; SC-560, 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole; i.g., intragastrically; TRPV1, transient receptor potential vanilloid type 1; RT-PCR, reverse transcriptase-polymerase chain reaction; 4-HNE, PG, prostaglandin; 4-HNE, trans-4-hydroxy-2-nonenal.
Address correspondence to: Dr. Tomasz Brzozowski, Department of Physiology, Jagiellonian University Medical College, 16 Grzegorzecka St., 31-531 Cracow, Poland. E-mail: mpbrzozo{at}cyf-kr.edu.pl
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