Roles of Calcitonin Gene–Related Peptide in Maintenance of Gastric Mucosal Integrity and in Enhancement of Ulcer Healing and Angiogenesis
2007; Elsevier BV; Volume: 134; Issue: 1 Linguagem: Inglês
10.1053/j.gastro.2007.10.001
ISSN1528-0012
AutoresTakashi Ohno, Youichiro Hattori, Rie Komine, Takako Ae, Sumito Mizuguchi, Katsuharu Arai, Takeo Saeki, Tatsunori Suzuki, Kanako Hosono, Izumi Hayashi, Yoshio Oh-hashi, Yukiko Kurihara, Hiroki Kurihara, Kikuko Amagase, Susumu Okabe, Katsunori Saigenji, Masataka Majima,
Tópico(s)Enhanced Recovery After Surgery
ResumoBackground & Aims: The gastrointestinal tract is known to be rich in neural systems, among which afferent neurons are reported to exhibit protective actions. We tested whether an endogenous neuropeptide, calcitonin gene–related peptide (CGRP), can prevent gastric mucosal injury elicited by ethanol and enhance healing of acetic acid–induced ulcer using CGRP knockout mice (CGRP−/−). Methods: The stomach was perfused with 1.6 mmol/L capsaicin or 1 mol/L NaCl, and gastric mucosal injury elicited by 50% ethanol was estimated. Levels of CGRP in the perfusate were determined by enzyme immunoassay. Gastric ulcers were induced by serosal application of absolute acetic acid. Results: Capsaicin inhibited injured area dose-dependently. Fifty percent ethanol containing capsaicin immediately increased intragastric levels of CGRP in wild-type (WT) mice, although 50% ethanol alone did not. The protective action of capsaicin against ethanol was completely abolished in CGRP−/−. Preperfusion with 1 mol/L NaCl increased CGRP release and reduced mucosal damage during ethanol perfusion. However, 1 mol/L NaCl was not effective in CGRP−/−. Healing of ulcer elicited by acetic acid in CGRP−/− mice was markedly delayed, compared with that in WT. In WT, granulation tissues were formed at the base of ulcers, and substantial neovascularization was induced, whereas those were poor in CGRP−/−. Expression of vascular endothelial growth factor was more markedly reduced in CGRP−/− than in WT. Conclusions: CGRP has a preventive action on gastric mucosal injury and a proangiogenic activity to enhance ulcer healing. These results indicate that the CGRP-dependent pathway is a good target for regulating gastric mucosal protection and maintaining gastric mucosal integrity. Background & Aims: The gastrointestinal tract is known to be rich in neural systems, among which afferent neurons are reported to exhibit protective actions. We tested whether an endogenous neuropeptide, calcitonin gene–related peptide (CGRP), can prevent gastric mucosal injury elicited by ethanol and enhance healing of acetic acid–induced ulcer using CGRP knockout mice (CGRP−/−). Methods: The stomach was perfused with 1.6 mmol/L capsaicin or 1 mol/L NaCl, and gastric mucosal injury elicited by 50% ethanol was estimated. Levels of CGRP in the perfusate were determined by enzyme immunoassay. Gastric ulcers were induced by serosal application of absolute acetic acid. Results: Capsaicin inhibited injured area dose-dependently. Fifty percent ethanol containing capsaicin immediately increased intragastric levels of CGRP in wild-type (WT) mice, although 50% ethanol alone did not. The protective action of capsaicin against ethanol was completely abolished in CGRP−/−. Preperfusion with 1 mol/L NaCl increased CGRP release and reduced mucosal damage during ethanol perfusion. However, 1 mol/L NaCl was not effective in CGRP−/−. Healing of ulcer elicited by acetic acid in CGRP−/− mice was markedly delayed, compared with that in WT. In WT, granulation tissues were formed at the base of ulcers, and substantial neovascularization was induced, whereas those were poor in CGRP−/−. Expression of vascular endothelial growth factor was more markedly reduced in CGRP−/− than in WT. Conclusions: CGRP has a preventive action on gastric mucosal injury and a proangiogenic activity to enhance ulcer healing. These results indicate that the CGRP-dependent pathway is a good target for regulating gastric mucosal protection and maintaining gastric mucosal integrity. Calcitonin gene–related peptide (CGRP), a 37-amino acid neuropeptide, has various biological actions, including responses to sensory stimuli, cardiovascular regulation, and vasodilation. Recent studies using genetically engineered mice have shown that CGRP-knockout mice exhibit increased blood pressure and overactivation of the sympathetic nervous system.1Oh-hashi Y. Shindo T. Kurihara Y. et al.Elevated sympathetic nervous activity in mice deficient in alphaCGRP.Circ Res. 2001; 89: 983-990Crossref PubMed Scopus (140) Google Scholar CGRP is synthesized by sensory C-fibers throughout the respiratory tree and potently constricts airway smooth muscle.2Pinto A. Sekizawa K. Yamaya M. et al.Effects of adrenomedullin and calcitonin gene–related peptide on airway and pulmonary vascular smooth muscle in guinea-pigs.Br J Pharmacol. 1996; 119: 1477-1483Crossref PubMed Scopus (38) Google Scholar Besides these actions, CGRP also exhibits significant roles in the gastrointestinal tract. Maintenance of gastric mucosal integrity is highly dependent on the alarm systems, which can rapidly sense the harmful chemical or mechanical stimuli exposed to the mucosa. The gastrointestinal tract is known to be rich in neural systems, among which afferent neurones of extrinsic origin are reported to operate as the emergency protective system.3Holzer P. Neural emergency system in the stomach.Gastroenterology. 1998; 114: 823-839Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar The functions of these afferents sensitive to chemicals are thought to be mediated by neuropeptides such as substance P and calcitonin gene–related peptide (CGRP) released from the C and Aδ fibers of sensory nerves in gastric mucosa.4Green T. Dockray G.J. Calcitonin gene-related peptide and substance P in afferents to the upper gastrointestinal tract in the rat.Neurosci Lett. 1987; 76: 151-156Crossref PubMed Scopus (147) Google Scholar, 5Sharkey K.A. Williams R.G. Dockray G.J. Sensory substance P innervation of the stomach and pancreas Demonstration of capsaicin-sensitive sensory neurons in the rat by combined immunohistochemistry and retrograde tracing.Gastroenterology. 1984; 87: 914-921PubMed Google Scholar It was reported by several groups including ours that CGRP protects against the gastric mucosal injury induced by ethanol and other substances.6Hayashi H. Ohno T. Nishiyama K. et al.Transient prevention of ethanol-induced gastric lesion by capsaicin due to release of endogenous calcitonin gene-related peptide in rats.Jpn J Pharmacol. 2001; 86: 351-354Crossref PubMed Scopus (16) Google Scholar, 7Boku K. Ohno T. Saeki T. et al.Adaptive cytoprotection mediated by prostaglandin I(2) is attributable to sensitization of CRGP-containing sensory nerves.Gastroenterology. 2001; 120: 134-143Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 8Arai K. Ohno T. Saeki T. et al.Endogenous prostaglandin I2 regulates the neural emergency system through release of calcitonin gene related peptide.Gut. 2003; 52: 1242-1249Crossref PubMed Scopus (32) Google Scholar Our series of intravital microscopic studies9Saeki T. Ohno T. Kamata K. et al.Mild irritant prevents ethanol-induced gastric mucosal microcirculatory disturbances through actions of calcitonin gene-related peptide and PGI2 in rats.Am J Physiol Gastrointest Liver Physiol. 2004; 286: G68-G75Crossref PubMed Scopus (28) Google Scholar, 10Ohno T. Katori M. Majima M. et al.Dilatation and constriction of rat gastric mucosal microvessels through prostaglandin EP2 and EP3 receptors.Aliment Pharmacol Ther. 1999; 13: 1243-1250Crossref PubMed Scopus (20) Google Scholar, 11Ohno T. Katori M. Nishiyama K. et al.Direct observation of microcirculation of the basal region of rat gastric mucosa.J Gastroenterol. 1995; 30: 557-564Crossref PubMed Scopus (30) Google Scholar revealed that ethanol-induced gastric mucosal injury was initially attributed to the congestion of mucosa caused by constrictions of collecting venules. CGRP prevented ethanol-induced constriction of the collecting venules and inhibited mucosal injury.12Ohno T. Katori M. Majima M. et al.Different responses of arterioles and venules in rat gastric mucosal microcirculation to endothelin-1 and endothelin-3.J Clin Gastroenterol. 1995; 21: S56-S65PubMed Google Scholar Prostaglandins (PGs) are known to contribute to the maintenance of gastric integrity. We recently reported that CGRP release was enhanced by endogenous PGs generated in the stomach under mild stimulation by 1 mol/L NaCl solution (1 mol/L NaCl)7Boku K. Ohno T. Saeki T. et al.Adaptive cytoprotection mediated by prostaglandin I(2) is attributable to sensitization of CRGP-containing sensory nerves.Gastroenterology. 2001; 120: 134-143Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar and that the CGRP released prevented gastric mucosal injury. But, despite intense studies, the evidence that the CGRP released from sensory nerves as an important preventive mediator exhibits gastric mucosal protective action has not been fully elucidated, because of the lack of adequate tools. Furthermore, we recently found that CGRP could enhance angiogenesis in vivo. Angiogenesis is involved in many physiologic and pathologic conditions, including the female reproductive cycle, embryonic development, tumor growth, metastasis, chronic inflammation, and retinopathy.13Ikeda Y. Hayashi I. Kamoshita E. et al.Host stromal bradykinin B2 receptor signaling facilitates tumor-associated angiogenesis and tumor growth.Cancer Res. 2004; 64: 5178-5185Crossref PubMed Scopus (79) Google Scholar, 14Majima M. Amano H. Hayashi I. Prostanoid receptor signaling relevant to tumor growth and angiogenesis.Trends Pharmacol Sci. 2003; 24: 524-529Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 15Katada J. Muramatsu M. Hayashi I. et al.Significance of vascular endothelial cell growth factor up-regulation mediated via a chymase-angiotensin-dependent pathway during angiogenesis in hamster sponge granulomas.J Pharmacol Exp Ther. 2002; 302: 949-956Crossref PubMed Scopus (34) Google Scholar It is widely accepted that the wound healing process is highly dependent on angiogenesis.16Kamoshita E. Ikeda Y. Fujita M. et al.Recruitment of a prostaglandin E receptor subtype, EP3-expressing bone marrow cells is crucial in wound-induced angiogenesis.Am J Pathol. 2006; 169: 1458-1472Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar The sponge implantation model used to evaluate angiogenesis showed that CGRP has a proangiogenic activity. This fact indicated that the angiogenesis-dependent ulcer healing processes may be enhanced by endogenous CGRP. CGRP may have a role not only in prevention of microcirculatory dysfunction but also in facilitation of gastric ulcer healing. To test these issues, we have developed a strain of knockout mice in which the genes for CGRP are disrupted.1Oh-hashi Y. Shindo T. Kurihara Y. et al.Elevated sympathetic nervous activity in mice deficient in alphaCGRP.Circ Res. 2001; 89: 983-990Crossref PubMed Scopus (140) Google Scholar The current study, using these mice, can therefore address the important roles of endogenous CGRP in the modulation of the neural emergency system and in the ulcer healing processes. The current study may open a new door to strategies for protecting gastric mucosal integrity and for treating gastric mucosal injury through CGRP-dependent mechanisms. A strain of 8-week-old male CGRP receptor knockout mice (CGRP−/−)1 and their wild-type counterparts (WT, male) were used. To estimate immunohistochemical localization of CGRP in gastric mucosa, we isolated gastric specimens under normal conditions from WT mice, and the sections were stained with rabbit polyclonal antibody against CGRP (Cambridge Research Biochemicals, Northwich, UK) as described earlier. All mice were kept in a room maintained at a constant temperature (25° ± 1°C) and humidity (60% ± 5%) throughout the experimental periods and were allowed free access to normal chow and water. The experiments were performed in accordance with the guidelines for animal experiments of Kitasato University School of Medicine. The experiments were performed on the animals after they had been anesthetized with urethane by intraperitoneal injection (1.225 g/kg; Aldrich Chemical Co, Milwaukee, WI). After laparotomy of the anesthetized CGRP−/− and WT mice, the stomach was doubly cannulated, from both esophageal and duodenal ends. Physiologic saline, 50% ethanol, or 1 mol/L NaCl or 50% ethanol containing 1.6 mmol/L capsaicin (37°C) was perfused at 1 mL/min, and the areas of the mucosal lesions were estimated by the method described previously.7Boku K. Ohno T. Saeki T. et al.Adaptive cytoprotection mediated by prostaglandin I(2) is attributable to sensitization of CRGP-containing sensory nerves.Gastroenterology. 2001; 120: 134-143Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 8Arai K. Ohno T. Saeki T. et al.Endogenous prostaglandin I2 regulates the neural emergency system through release of calcitonin gene related peptide.Gut. 2003; 52: 1242-1249Crossref PubMed Scopus (32) Google Scholar The exposure periods for 50% ethanol, 1 mol/L NaCl, and 50% ethanol containing 1.6 mmol/L capsaicin were 2, 4, and 4 minutes, respectively.7Boku K. Ohno T. Saeki T. et al.Adaptive cytoprotection mediated by prostaglandin I(2) is attributable to sensitization of CRGP-containing sensory nerves.Gastroenterology. 2001; 120: 134-143Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 8Arai K. Ohno T. Saeki T. et al.Endogenous prostaglandin I2 regulates the neural emergency system through release of calcitonin gene related peptide.Gut. 2003; 52: 1242-1249Crossref PubMed Scopus (32) Google Scholar CGRP levels in the perfusate from anesthetized mice were determined by the method described in our previous report.8Arai K. Ohno T. Saeki T. et al.Endogenous prostaglandin I2 regulates the neural emergency system through release of calcitonin gene related peptide.Gut. 2003; 52: 1242-1249Crossref PubMed Scopus (32) Google Scholar The contents of CGRP in CGRP−/− and in WT were also determined by enzyme immunoassay after extraction of CGRP from the tissues of the stomach. Under ether anesthesia, the stomachs were isolated and rapidly frozen in liquid nitrogen. The frozen stomach was pulverized in a stainless steel cylinder cooled with liquid nitrogen. Powders thus produced were added to 3 mL of 2 N acetic acid, and the resulting mixtures were bathed in 90°C water for 20 minutes and then centrifuged at 4500g for 10 minutes (4°C). CGRP was extracted from the supernatant using reverse-phase C18 columns (Amersham, Little Chalfont, UK). The supernatant was applied to the column, followed by washing with 20 mL of 0.1% trifluoroacetic acid. CGRP was eluted with 3 mL of 60% acetonitrile in 0.1% trifluoroacetic acid and was determined by an enzyme immunoassay. Results are expressed as picograms of CGRP per gram of wet tissue of the stomach. The levels of prostaglandin E2 and 6-keto-prostaglandin F1α in the perfusates from anesthetized mice were measured as in our previous report.7Boku K. Ohno T. Saeki T. et al.Adaptive cytoprotection mediated by prostaglandin I(2) is attributable to sensitization of CRGP-containing sensory nerves.Gastroenterology. 2001; 120: 134-143Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 8Arai K. Ohno T. Saeki T. et al.Endogenous prostaglandin I2 regulates the neural emergency system through release of calcitonin gene related peptide.Gut. 2003; 52: 1242-1249Crossref PubMed Scopus (32) Google Scholar Experiments on tube formation were conducted in triplicate in 24-multiwell dishes using Angiogenesis kit (Kurabo, Okayama, Japan), according to manufacturer's instructions. Human umbilical vein endothelial cells (HUVECs) cocultured with human fibroblasts were treated with the medium containing CGRP (final concentrations, 3 μmol/L and 30 μmol/L) for 11 days. The medium containing CGRP was changed every 3 days. After 11 days, dishes were washed with phosphate-buffered saline and fixed with 70% ethanol at 41°C. After the fixed cells were rinsed 3 times with phosphate-buffered saline, cells were stained with mouse antihuman CD31 (Kurabo). Finally, the cells were viewed in a microscope with a digital camera. The tube length was measured quantitatively with the Kurabo angiogenesis image analyzer (Kurabo) in 5 different fields per well, and the mean values were analyzed statistically. Circular sponge discs were then implanted into subcutaneous tissues of the back of WT, as described previously.17Amano H. Hayashi I. Endo H. et al.Host prostaglandin E(2)-EP3 signaling regulates tumor-associated angiogenesis and tumor growth.J Exp Med. 2003; 197: 221-232Crossref PubMed Scopus (287) Google Scholar, 18Majima M. Hayashi I. Muramatsu M. et al.Cyclooxygenase-2 enhances basic fibroblast growth factor-induced angiogenesis through the induction of vascular endothelial growth factor in rat sponge implants.Br J Pharmacol. 2000; 130: 641-649Crossref PubMed Scopus (186) Google Scholar, 19Majima M. Isono M. Ikeda Y. et al.Significant roles of inducible cyclooxygenase (COX)-2 in angiogenesis in rat sponge implants.Jpn J Pharmacol. 1997; 75: 105-114Crossref PubMed Scopus (99) Google Scholar Neovascularization was assessed by measuring the concentration of hemoglobin in the granulation tissues around sponge implants. To enhance the neovascularization of the sponge implants, CGRP was injected daily (for 1 week, 10 mg/sponge/day) directly into the sponge implants using a 26G needle under light ether anesthesia. In a separate experiment, basic fibroblast growth factor (bFGF) was topically injected for a week (1 nmol/sponge/day). Furthermore, a CGRP antagonist, CGRP (8-37) (100 nmol/sponge/day) was topically injected into some sponges for 7 days. Acetic acid (purity 99.7%, Wako, Osaka, Japan) was used to elicit the gastric ulcer. Mice were anesthetized with ether. Gastric ulcers were induced by brief serosal application of acetic acid, according to a previous report made by us.20Okabe S. Amagase K. An overview of acetic acid ulcer models—the history and state of the art of peptic ulcer research.Biol Pharm Bull. 2005; 28: 1321-1341Crossref PubMed Scopus (194) Google Scholar The animals were fed normally thereafter. The stomachs were removed, and the ulcerated area (mm2) was measured.20Okabe S. Amagase K. An overview of acetic acid ulcer models—the history and state of the art of peptic ulcer research.Biol Pharm Bull. 2005; 28: 1321-1341Crossref PubMed Scopus (194) Google Scholar The area of each ulcer was determined under a dissecting microscope with a square grid (Olympus, Tokyo, Japan). Microvessel density in the ulcer granulation tissues was assessed as a parameter of angiogenesis according to the established methods described previously.17Amano H. Hayashi I. Endo H. et al.Host prostaglandin E(2)-EP3 signaling regulates tumor-associated angiogenesis and tumor growth.J Exp Med. 2003; 197: 221-232Crossref PubMed Scopus (287) Google Scholar CD-31 immunoreactive endothelial cells were stained with CD31 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and were differentiated from other connective tissue elements. Microvessel density was expressed in terms of the number of microvessels per observed area (vessel number/mm2). Transcripts encoding CRLR (a subunit of CGRP1), CTRa (a subunit of CGRP2), RAMP1 (a common subunit of CGRP receptor), cyclooxygenase (COX)-1, COX-2, isoforms of nitric oxide synthases (eNOS, nNOS, and iNOS), VEGF-A, CD31, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined by reverse transcriptase–polymerase chain reaction (RT-PCR) analysis, as described previously.17Amano H. Hayashi I. Endo H. et al.Host prostaglandin E(2)-EP3 signaling regulates tumor-associated angiogenesis and tumor growth.J Exp Med. 2003; 197: 221-232Crossref PubMed Scopus (287) Google Scholar The amplification protocol comprised 28 cycles (COX-1, COX-2, CD31), 31 cycles (CRLR, CTRa, RAMP1, VEGF-A), or 23 cycles (GAPDH) of 45 seconds at 94°C, 60 seconds at 55°C, and 60 seconds at 72°C. The reaction mixtures were subsequently applied to a 2% agarose gel, and the amplified products were stained with ethidium bromide. Primers used were as follows: 5′-CTTCTGGATGCTCTGTGAAGG-3′2 (sense) and 5′2-CCCAGCCGAGAAAATAATACC-3′2 (antisense) for CRLR, 5′2-GAACTGTCACCACCCTTACCC-3′ (sense) and 5′-TCGCAGAGCATCCAGAAGTAG-3′ (antisense) for CTRa, 5′-CCATCTCTTCATGGTCACTGC-3′ (sense) and 5′-AGCGTCTTCCCAATAGTCTCC-3′ (antisense) for RAMP1, 5′-TCCCTGAGATCTGGACCTGGC-3′ (sense) and 5′-TGAGTACTTCTCGGATGAAGG-3 (antisense) for COX-1, 5′-TGGGTGTGAAGGGAAATAAGG-3′ (sense) and 5′-CATCATATTTGAGCCTTGGGG-3′ (antisense) for COX-2, 5'-AACCATGAACTTTCTGCTCTC-3' (sense) and 5'-GTGATTTTCTGGCTTTGTTC-3' (antisense) for VEGF, 5'-CGGGATCCAGGAAAGCCAAGGCCAAA-3' (sense) and 5'-CGGAAT-TCTTGACTGTCTTAAGTTCC-3' (antisense) for CD31, and 5'-CCCTTCATTGACCTCAACTACAATGGT-3' (sense) and 5'-GAGGGGCCATCCACAGTCTTCTG-3' (antisense) for GAPDH. Semiquantitative RT-PCR for CD31 and VEGF was performed according to the method described previously.15Katada J. Muramatsu M. Hayashi I. et al.Significance of vascular endothelial cell growth factor up-regulation mediated via a chymase-angiotensin-dependent pathway during angiogenesis in hamster sponge granulomas.J Pharmacol Exp Ther. 2002; 302: 949-956Crossref PubMed Scopus (34) Google Scholar Expression of GAPDH mRNA was used as an internal control, and the VEGF mRNA or CD31 mRNA expression level was presented as the ratio of VEGF mRNA or CD31 mRNA to GAPDH mRNA expressed in the ulcer granulation tissues. Normal gastric mucosal tissues without ulceration were also examined. After perfusion with 50% ethanol containing capsaicin, the glandular stomach was isolated and was rapidly frozen in liquid nitrogen. The frozen tissue was pulverized, and total RNA was extracted from the pulverized tissue with Isogen. RT-PCR for eNOS, nNOS, iNOS, and GAPDH was performed as described previously. The amplification protocol comprised 28 cycles (eNOS, nNOS, iNOS) and 23 cycles (GAPDH) of 45 seconds at 94°C, 60 seconds at 55°C, and 60 seconds at 72°C. The primers used were as follows: 5′-GCCTAACTCCTGTCTTCCATC-3′ (sense) and 5′-TTCACTGCATTGGCTACTTCC-3′ (antisense) for eNOS, 5′-GCTGTGCTTTGATGGAGATGA-3′ (sense) and 5′-ACTTGCGGGAGTCAGAATAGG-3′ (antisense) for nNOS, and 5′-AAAACCCCTTGTGCTGTTCTC-3′ (sense) and 5′-CTGGAACATTCTGTGCTGTCC-3′ (antisense) for iNOS. Data are expressed as means ± SEM. Comparisons among multiple groups were performed by factorial analysis of variance (ANOVA) followed by Scheffe's test. Comparisons between 2 groups were performed with Student t test. A P value of <.05 was considered statistically significant. In WT, immunoreactive CGRP was present in the nerve fibers of the gastric mucosa (Figure 1A) and submucosal layer (Figure 1B), whereas the immunoreactive CGRP was hardly detected in the gastric mucosa in CGRP−/− (data not shown). When CGRP contents in the stomach were determined by enzyme immunoassay in WT, CGRP detected was 13.0 ± 2.1 pg/mg wet tissue of stomach (Figure 1C). By contrast, CGRP content in CGRP−/− was below the detection limits (0.05 pg/mg wet tissue of stomach) (Figure 1C). As the left panel in Figure 2A shows, 2-minute exposure of the gastric mucosa to 50% ethanol in WT resulted in mucosal injury, which was recognized as a reddened area and covered approximately 20% of the glandular stomach. The gastric lesion by ethanol was markedly suppressed by coexposure of gastric mucosa to capsaicin (1.6 mmol/L) in 50% ethanol (Figure 2A, right panel). Perfusion with 50% ethanol after physiologic saline resulted in gastric mucosal lesions (19.7% ± 0.9%, n = 5) in WT (Figure 2B). Simultaneous application of capsaicin to ethanol reduced gastric lesions in a dose-dependent manner (Figure 2B). When intragastric level of CGRP was determined, the resting level of CGRP was 2 pg per stomach per 4 minutes, and exposure of the stomach to ethanol did not increase CGRP levels (Figure 2C). By contrast, administration of capsaicin (1.6 mmol/L) with ethanol markedly increased CGRP levels (Figure 2C). In CGRP−/−, administration of capsaicin (1.6 mmol/L) with 50% ethanol did not show any preventive actions against ethanol, which is shown in WT (Figure 2D). These results suggest that endogenous CGRP released by capsaicin certainly suppressed the ethanol-induced gastric mucosal injury. Because we had previously reported that endogenous prostaglandin I2 facilitated the release of CGRP, we determined endogenous prostaglandin levels during perfusion experiment (Table 1). Resting intragastric 6-keto-prostaglandin F1α levels were about 30 pg per stomach per 4 minutes both in WT and in CGRP−/−, and the exposure to 50% ethanol containing capsaicin significantly increased the intragastric 6-keto-prostaglandin F1α levels to about threefold in the first perfusate after exposure (Table 1). The increased levels were not different between WT and in CGRP−/−. When the perfusate was again replaced with physiologic saline, the intragastric 6-keto-prostaglandin F1α levels were reduced during 4 minutes of perfusion in WT and in CGRP−/−.Table 1Intragastric Prostaglandin Levels During Perfusion ExperimentsPGsMouse (n = 5)Saline beforeCapsaicin + 50% ethanolSaline after Capsaicin + ethanolpg/4 min6-ketoWT25.2 ± 3.689.6 ± 9.6aP < .05.36.3 ± 4.1PGF1αCGRP−/−36.0 ± 3.998.6 ± 8.9aP < .05.49.7 ± 4.6WT26.7 ± 3.535.7 ± 3.629.3 ± 3.1PGE2CGRP−/−36.2 ± 4.640.8 ± 4.436.6 ± 4.2Note. Perfusion was performed in wild-type mice (C57BL/6) and CGRP knockout mice according to the protocol described in Materials and Methods. The levels of 6-keto-prostaglandin F1α (PGF1α) and prostaglandin E2 (PGE2) in the perfusates were determined in each 4-min sample.Values (pg/4 min) are expressed as means ± SEM from 5 mice. ANOVA was used for statistical analysis. The values during the perfusion of capsaicin + ethanol were compared with the basal control values (saline).a P < .05. Open table in a new tab Note. Perfusion was performed in wild-type mice (C57BL/6) and CGRP knockout mice according to the protocol described in Materials and Methods. The levels of 6-keto-prostaglandin F1α (PGF1α) and prostaglandin E2 (PGE2) in the perfusates were determined in each 4-min sample. Values (pg/4 min) are expressed as means ± SEM from 5 mice. ANOVA was used for statistical analysis. The values during the perfusion of capsaicin + ethanol were compared with the basal control values (saline). The basal resting levels of intragastric prostaglandin E2 during the perfusion of physiologic saline were also very low in WT and CGRP−/−. The levels were not increased in WT and in CGRP−/−, even after the intragastric application of 50% ethanol containing capsaicin (Table 1). Furthermore, we tested the expression of nitric oxide synthase isoforms in the stomach after exposure of 50% ethanol containing capsaicin. We had detected the expression of eNOS, nNOS, and iNOS in the extracts of the glandular stomach isolated from WT (Figure 2E). The intensity of the expressions of the NOS isoforms in CGRP−/− mice was not different from that in WT (Figure 2E). Prior administration of 1 mol/L NaCl to the gastric mucosa for 4 minutes reduced mucosal injury elicited by 50% ethanol in WT (Figure 3A). This NaCl effect was observed in a concentration-dependent manner (Figure 3B). Intragastric levels of CGRP were not increased by 50% ethanol alone from the basal values after addition of physiologic saline (Figure 3C, open circles). Before administration of 1 mol/L NaCl did not increase CGRP levels (Figure 3C, closed circles). By contrast, 50% ethanol administration after 1 mol/L NaCl markedly increased the CGRP levels (Figure 3C, closed circles). These findings suggest that pre-exposure of 1 mol/L NaCl solution facilitates CGRP release when ethanol is given and that increased CGRP may prevent ethanol-induced mucosal injury. This possibility was tested with the use of CGRP−/−. Figure 3D depicts ethanol-induced gastric mucosal lesions in WT and CGRP−/− after 1 mol/L NaCl treatment. In CGRP−/−, 1 mol/L NaCl administration before 50% ethanol did not show any preventive actions against ethanol, which was seen in WT (Figure 3D). To investigate the biologic effects of CGRP on angiogenesis in vitro, the tube formation assay was performed using HUVECs cocultured with fibroblasts. When HUVECs were cultured in the presence of CGRP, substantial tube formation was observed (Figure 4A). When the total length of the formed tubes was determined, the effect of CGRP at final concentrations of 3 μmol/L and 30 μmol/L was statistically significant (Figure 4B). Daily topical injections of CGRP to the sponge implants increased the redness of the granulation tissues (Figure 4C) and the hemoglobin contents in these tissues (Figure 4D). These results indicate that CGRP exhibits proangiogenic activity in vivo. Topical injection of bFGF to the sponges markedly increased angiogenesis fourfold. This enhanced angiogenesis was significantly reduced with the concomitant administration of CGRP1 antagonist CGRP (8-37) (Figure 4E). This suggests that endogenous CGRP has a proangiogenic activity in vivo. A brief serosal administration of 100% acetic acid caused gastric ulcers that remained for more than 1 week. In WT, 7 days after acetic acid administration, ulcer size was less than 10% of the original size (upper panels, Figure 5A). By contrast, ulcer healing in CGRP−/− mice was markedly delayed (lower panels, Figure 5A). Figure 5B summarizes the changes of ulcer area in CGRP−/− and WT. Ulcer area in WT was gradually reduced during 2 weeks of the observation period; in contrast, that in CGRP−/− remained high. From day 3 to day 14, the healing process was significantly delayed in CGRP−/− compared with WT. Figure 6A shows typical hematoxylin and eosin staining results in acetic acid–induced ulcers at day 3. In WT, granulation tissues were formed at the base of the ulcers, and substantial neovascularization occurred, as indicated by the arrows. In contrast, the granulation tissue formation and angiogenesis were poor in CGRP−/−. Immunohistochemistry using CD31 antibody showed that the microvessel density in the ulcer granulation tissues in CGRP−/− at day 3
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