Capsaicin (3.3 mmol/l)

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1 A Vehicle BCTC (.8 mmol/l) Sensory deafferentation Time (min) Capsaicin (3.3 mmol/l) N=3 4 * P<.5 ***** ***** B Maximal GMBF Response Vehicle N=3 4 * P<.5 * * BCTC (.8 mm) Sensory deafferentation Capsaicin (3.3 mmol/l) Supplementary figure 1

2 Figure legends for supplementary figures Supplementary figure 1. Effects of BCTC, a TRPV1 antagonist, and sensory deafferentation on gastric mucosal blood flow (GMBF) induced by mucosal application of capsaicin in the ex vivo stomach of anesthetized rats. Chemical deafferentation (capsaicin pretreatment) was performed 2 weeks before the experiment by consecutive injections of capsaicin s.c. once daily for 3 days (total dose: mg/kg). Figure A shows the time course of GMBF response to capsaicin (3.3 mmol/l) in anesthetized rats treated with BCTC or sensory deafferentation. The stomach was perfused with saline, BCTC (.8 mmol/l) was applied to the chamber for 3 min, starting at min before the capsaicin application. Capsaicin was topically applied to the mucosa for 1 min. The data are expressed as % increase of basal values and represent the means±s.e. of values every 2 or 1 min from 3 4 rats. Statistically significant difference at P<.5: *from the corresponding values in the group treated with vehicle. Figure B shows a maximal GMBF response induced by mucosal application of capsaicin (3.3 mmol/l). The data are expressed as % increase of basal values and represent the means±s.e. from 3 4 rats. Statistically significant difference at P<.5: *from the group treated with vehicle. Note that increased GMBF in response to capsaicin was completely abolished by BCTC and sensory deafferentation. 1

3 A B Vehicle 14W (1 mg/kg) Time (min) Capsaicin (3.3 mmol/l) N= Maximal GMBF Response N=4 5 Vehicle W (mg/kg) Capsaicin (3.3 mmol/l) Supplementary figure 2

4 A Flow() (Arbitary Unit: mv) Vehicle (Saline, iv) saline(.3ml iv) 1:4 5: 58: 1:6:4 1:15: Capsapcin(3.3mM) Arterial 血圧 Pressure (mmhg) (mmhg) Vehicle (Saline, iv) saline(.3ml iv) Capsapcin(3.3mM) 1:4 5: 58: 1:6:4 1:15: B Gastric Mucosal Flow() Blood Flow (Arbitary Unit: mv) L-NAME (1 mg/kg, iv) L-NAME1mgkg, iv 1:15: 1:23: 1:31:4 1:4: 1:48: 1:56:4 Cap3.3mM 血圧 ( mhg) Arterial Pressure (mmhg) L-NAME (1 mg/kg, iv) L-NAME1mgkg, iv 1:15: 1:23: 1:31:4 1:4: 1:48: 1:56:4 Cap3.3mM Supplementary figure 3

5 Supplementary figures 2. Effect of 14W, a selective inos inhibitor, on gastric mucosal blood flow (GMBF) induced by capsaicin in the ex vivo stomach of anesthetized rats. Figure A shows the time course of analysis for the effect of 14W (1 mg/kg) on GMBF response to capsaicin (3.3 mmol/l) in anesthetized rats. The stomach was perfused with saline before the application, 14W (3 and 1 mg/kg) was given via intravenous injection min before capsaicin application, and capsaicin (3.3 mmol/l) was topically applied to the mucosa for 1 min. The data are expressed as % increase of basal values and represent the means±s.e. of values every 2 or 1 min from 4 rats. Figure B shows the maximal response of GMBF response during capsaicin (3.3 mmol/l) application in animals treated with 14W (3 and 1 mg/kg). The data are expressed as % increase of basal values and represent the means±s.e. from 4-5 rats. Note that increased GMBF in response to capsaicin was not affected by 14W. Supplementary figure 3. Representative effects of L-NAME, NPLA, and L-NIO on gastric mucosal blood flow (GMBF) and arterial pressure (AP) in urethane-anesthetized rats. Vehicle (saline; A), L-NAME (1 mg/kg; B), NPLA (2 mg/kg; C), and L-NIO (1 mg/kg; D) were intravenously injected min before capsaicin application. Note that AP and GMBF were obviously increased immediately after the administration of L-NAME (1 mg/kg) as compared with vehicle, and then AP, but not GMBF, was maintained at high levels throughout the experiment. In contrast, AP in the NPLA (2 mg/kg)-treated rats returned to the baseline, while AP, but not GMBF, was temporary increased after the administration of NPLA. AP in rats treated with L-NIO (1 mg/kg) was maintained at a level slightly above the baseline, although AP and GMBF were transiently increased after the administration of L-NIO. 2

6 C Flow() (Arbitary Unit: mv) NPLA (2 mg/kg, iv) NPLA(2mg/kg, iv) 1:48: 1:56:4 2:5: 2:13: 2:21:4 Capsaicin(3.3mM) Arterial 血圧 Pressure ( mhg) (mmhg) NPLA (2 mg/kg, iv) NPLA(2mg/kg, iv) 1:48: 1:56:4 2:5: 2:13: 2:21:4 Capsaicin(3.3mM) D Gastric Mucosal Flow() Blood Flow (Arbitary Unit: mv) L-NIO (1 mg/kg, iv) 1mg/kgL-NIO.28ml i.v. 25: 33: 41:4 5: 58:.1mg/kgBethanechol.28ml i.v. Bethanechol (5 g/kg/min, iv) Bethanechol (5 g/kg/min, iv) Arterial 血圧 Pressure ( mhg) (mmhg) L-NIO (1 mg/kg, iv) 1mg/kgL-NIO.28ml i.v. 25: 33: 41:4 5: 58:.1mg/kgBethanechol.28ml i.v. Supplementary figure 3

7 A B Bethanechol (iv-infusion) Bethanechol (2.5 g/kg/min) Bethanechol (5 g/kg/min) Time (min) n= Bethanechol (5 g/kg/min, iv) Vehicle L-NIO (1 mg/kg) n= Time (min) Supplementary figure 4

8 Supplementary figure 4. Inhibitory effect of the enos selective inhibitor L-NIO on bethanecholinduced on gastric mucosal blood flow (GMBF) in anesthetized rats. Figure A shows the time course of analysis for the effect of a non-selective muscarinic agonist bethanechol (2.5 and 5 g/kg/min) on GMBF response in anesthetized rats. Bethanechol (2.5 and 5 g/kg/min) was intravenous infused for min. The data are expressed as % increase of basal values and represent the means±s.e. of values every 2 or 1 min from 2 7 rats. Note that bethanechol (2.5 and 5 g/kg/min) dose-dependently increased GMBF. Because the effect of bethanechol on GMBF reached the maximal value during infusion at 5 g/kg/min, this dose was used in the following experiments. Figure B shows the time course of analysis for the inhibitory effect of L-NIO on bethanechol (5 g/kg/min)-induced GMBF response in anesthetized rats. L-NIO (1 mg/kg) was injected i.v. min, and bethanechol (5 g/kg/min) was intravenous infused for min. The data are expressed as % increase of basal values and represent the means±s.e. of values every 2 or 1 min from 2 7 rats. Note that the pretreatment of L-NIO inhibited the gastric mucosal hyperemia in response to bethanechol, although L-NIO (1 mg/kg, iv) was given min before the i.v. infusion of bethanechol. These data suggest that L-NIO (1 mg/kg, iv) inhibits enos/no production, resulting in the less vasodilation in response to bethanechol in the rat stomach. 3

9 A B C Contraction (% of ACh response) Contraction (% of ACh response) Contraction (% of ACh response) N=4 6 * P<.5 Vehicle N=3 4 * P<.5 * + L Arg (3 mm) # + D Arg (3 mm) L NAME ( M) AITC ( M) Vehicle 2 NPLA ( M) AITC ( M) N=3 6 * P<.5 Vehicle 1 3 L NIO ( M) AITC ( M) * * * Supplementary figure 5

10 Supplementary figure 5. Effects of treatment with N w -nitro-l-arginine methyl ester (L-NAME) and the combined treatment with L-arginine (L-Arg) or D-arginine (D-Arg) plus L-NAME on contractile responses to allyl isothiocyanate (AITC) in the isolated mouse distal colon (A). L- NAME ( M) was added 8 min before AITC ( M) was applied to preparations of the distal colon. Results are expressed as a percentage of the contractile responses induced by acetylcholine (ACh, 1 M) in each tissue. Data represent means±s.e. obtained from 4 6 experiments. The asterisk (*) denotes a value that is significantly different from isolated preparations treated with vehicle alone (P<.5, Bonferroni test). Note that contractile response to AITC in the distal colon was significantly enhanced by L-NAME, which was reversed by L-Arg. Effect of N w -propyl-l-arginine (NPLA), a selective inhibitor of neuronal NOS, on contractile responses to AITC in the isolated mouse rectum and distal colon (B). NPLA (.2- M) was added 8 min before application of AITC ( M) in each preparation of the distal colon. Results are expressed as a percentage of the contractile responses induced by acetylcholine (ACh, 1 M) in each tissue. Data represent means±s.e. obtained from 3 4 experiments. An asterisk (*) denotes a value that is significantly different from isolated preparations treated with vehicle alone (P<.5, Bonferroni test). Note that the contractile response to AITC in the distal colon was enhanced by NPLA, a selective inhibitor of neuronal NOS, in a dose dependent manner. Effect of N 5 -(1-iminoethyl)-L-ornithine (L-NIO), a selective inhibitor of endothelial NOS, on contractile responses to AITC in the isolated mouse distal colon (C). L-NIO (1- M) was added, and AITC ( M) was applied to preparations of the distal colon after 8 min. Results are expressed as a percentage of the contractile responses induced by acetylcholine (ACh, 1 M) in each tissue. Data represent means±s.e. obtained from 3 6 experiments. The asterisk (*) denotes a value that is significantly different from isolated preparations tread with vehicle alone (P<.5, Bonferroni test). Note that AITC-induced contraction in distal colon was significantly potentiated by a high dose of L-NIO ( M), but by not a low dose (<3 M). 4

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