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Traini C, Idrizaj E, Garella R, Squecco R, Vannucchi MG, Baccari MC. Glucagon-like peptide-2 interferes with the neurally-induced relaxant responses in the mouse gastric strips through VIP release. Neuropeptides 2020; 81:102031. [PMID: 32143816 DOI: 10.1016/j.npep.2020.102031] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/27/2019] [Revised: 01/20/2020] [Accepted: 02/16/2020] [Indexed: 02/07/2023]
Abstract
Glucagon-like peptide-2 (GLP-2) has been reported to indirectly relax gastric smooth muscle. In the present study we investigated, through a combined mechanical and immunohistochemical approach, whether GLP-2 interferes with the electrical field stimulation (EFS)-induced vipergic relaxant responses and the mechanism through which it occurs. For functional experiments, strips from the mouse gastric fundus were mounted in organ baths for isometric recording of the mechanical activity. Vasoactive intestinal peptide (VIP) immunoreactivity in GLP-2 exposed specimens was also evaluated by immunohistochemistry. In carbachol pre-contracted strips, GLP-2 (20 nM) evoked a tetrodotoxin (TTX)-sensitive relaxation, similar in shape to the TTX-insensitive of 100 nM VIP. In the presence of GLP-2, VIP had no longer effects and no more response to GLP-2 was observed following VIP receptor saturation. EFS (4-16 Hz) induced a fast relaxant response followed, at the higher stimulation frequencies (≥ 8 Hz), by a slow one. This latter was abolished either by GLP-2 or VIP receptor saturation as well as by the VIP receptor antagonist, VIP 6-28 (10 μM). A decrease of VIP-immunoreactive nerve structures in the GLP-2 exposed specimens was observed. These results suggest that, in the mouse gastric fundus, GLP-2 influences the EFS-induced slow relaxant response by promoting neuronal VIP release.
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Affiliation(s)
- Chiara Traini
- Department of Experimental and Clinical Medicine, Histology and Embryology Research Unit, University of Florence, 50134 Florence, Italy
| | - Eglantina Idrizaj
- Department of Experimental and Clinical Medicine, Section of Physiological Sciences, University of Florence, 50134 Florence, Italy
| | - Rachele Garella
- Department of Experimental and Clinical Medicine, Section of Physiological Sciences, University of Florence, 50134 Florence, Italy
| | - Roberta Squecco
- Department of Experimental and Clinical Medicine, Section of Physiological Sciences, University of Florence, 50134 Florence, Italy
| | - Maria Giuliana Vannucchi
- Department of Experimental and Clinical Medicine, Histology and Embryology Research Unit, University of Florence, 50134 Florence, Italy
| | - Maria Caterina Baccari
- Department of Experimental and Clinical Medicine, Section of Physiological Sciences, University of Florence, 50134 Florence, Italy.
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Min YW, Ko EJ, Lee JY, Rhee PL. Impaired neural pathway in gastric muscles of patients with diabetes. Sci Rep 2018; 8:7101. [PMID: 29739973 PMCID: PMC5940896 DOI: 10.1038/s41598-018-24147-y] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2018] [Accepted: 03/22/2018] [Indexed: 12/22/2022] Open
Abstract
To explore the pathogenic mechanism of diabetic gastropathy, we investigated differences in response to electrical field stimulation (EFS) of gastric muscles from diabetic and non-diabetic (control) patients. Gastric specimens were obtained from 34 patients and 45 controls who underwent gastrectomy for early gastric cancer. Using organ bath techniques, we examined peak and nadir values of contraction under EFS. To examine responses to purinergic and nitrergic inhibition without cholinergic innervation, atropine, MRS2500, and N-nitro-L-arginine (L-NNA) were added sequentially to the organ bath. Tetrodotoxin (TTX) was used to confirm that the responses to EFS were mediated via neural stimulation. In the absence of pharmacological agents, peak contraction amplitude was greater in non-diabetic controls compared to diabetics only in the distal longitudinal gastric muscles. However, the nadir was greater in controls than in patients in both proximal and distal gastric circular muscles. Addition of MRS2500 could not decrease the nadir in both controls and patients, both in the proximal and distal stomach. However, L-NNA completely reversed the relaxation. TTX had no further effect on nadir. In conclusion, impaired inhibitory nitrergic neural pathway in both proximal and distal stomach and impaired excitatory cholinergic neural pathway in the distal stomach may contribute to the pathogenic mechanism underlying diabetic gastropathy.
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Affiliation(s)
- Yang Won Min
- Department of Medicine, Sungkyunkwan University School of Medicine, Seoul, Korea
| | - Eun-Ju Ko
- Biomedical Research Institute, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
| | - Ji-Yeon Lee
- Biomedical Research Institute, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
| | - Poong-Lyul Rhee
- Department of Medicine, Sungkyunkwan University School of Medicine, Seoul, Korea.
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Garella R, Idrizaj E, Traini C, Squecco R, Vannucchi MG, Baccari MC. Glucagon-like peptide-2 modulates the nitrergic neurotransmission in strips from the mouse gastric fundus. World J Gastroenterol 2017; 23:7211-7220. [PMID: 29142468 PMCID: PMC5677198 DOI: 10.3748/wjg.v23.i40.7211] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/07/2017] [Revised: 09/19/2017] [Accepted: 09/26/2017] [Indexed: 02/06/2023] Open
Abstract
AIM To investigate whether glucagon-like peptide-2 (GLP-2) influences the neurally-induced responses in gastric strips from mice, since no data are available.
METHODS For functional experiments, gastric fundal strips were mounted in organ baths containing Krebs-Henseleit solution. Mechanical responses were recorded via force-displacement transducers, which were coupled to a polygraph for continuous recording of isometric tension. Electrical field stimulation (EFS) was applied via two platinum wire rings through which the preparation was threaded. The effects of GLP-2 (2 and 20 nmol/L) were evaluated on the neurally-induced contractile and relaxant responses elicited by EFS. Neuronal nitric oxide synthase (nNOS) enzyme was evaluated by immunohistochemistry.
RESULTS In the functional experiments, electrical field stimulation (EFS, 4-16 Hz) induced tetrodotoxin (TTX)-sensitive contractile responses, which were reduced in amplitude by GLP-2 (P < 0.05). In the presence of the nitric oxide (NO) synthesis inhibitor L-NNA, GLP-2 no longer influenced the neurally-evoked contractile responses (P > 0.05). The direct smooth muscle response to methacholine was not influenced by GLP-2 (P > 0.05). In the presence of guanethidine and carbachol, the addition of GLP-2 to the bath medium evoked TTX-sensitive relaxant responses that were unaffected by L-NNA (P > 0.05). EFS induced a fast NO-mediated relaxation, whose amplitude was enhanced in the presence of the hormone (P < 0.05). Immunohistochemical experiments showed a significant increase (P < 0.05) in nNOS immunoreactivity in the nerve structures after GLP-2 exposure.
CONCLUSION The results demonstrate that in gastric fundal strips, GLP-2 influences the amplitude of neurally-induced responses through the modulation of the nitrergic neurotransmission and increases nNOS expression.
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Affiliation(s)
- Rachele Garella
- Department of Experimental and Clinical Medicine, Section of Physiology, University of Florence, 50134 Florence, Italy
| | - Eglantina Idrizaj
- Department of Experimental and Clinical Medicine, Section of Physiology, University of Florence, 50134 Florence, Italy
| | - Chiara Traini
- Department of Experimental and Clinical Medicine, Histology and Embryology Research Unit, University of Florence, 50134 Florence, Italy
| | - Roberta Squecco
- Department of Experimental and Clinical Medicine, Section of Physiology, University of Florence, 50134 Florence, Italy
| | - Maria Giuliana Vannucchi
- Department of Experimental and Clinical Medicine, Histology and Embryology Research Unit, University of Florence, 50134 Florence, Italy
| | - Maria Caterina Baccari
- Department of Experimental and Clinical Medicine, Section of Physiology, University of Florence, 50134 Florence, Italy
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Fujimoto Y, Hayashi S, Azuma YT, Mukai K, Nishiyama K, Kita S, Morioka A, Nakajima H, Iwamoto T, Takeuchi T. Overexpression of Na +/Ca 2+ exchanger 1 display enhanced relaxation in the gastric fundus. J Pharmacol Sci 2016; 132:181-186. [PMID: 27816547 DOI: 10.1016/j.jphs.2016.10.003] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2016] [Revised: 10/06/2016] [Accepted: 10/11/2016] [Indexed: 02/08/2023] Open
Abstract
In gastric smooth muscles, the released Ca2+ activates the contractile proteins and Ca2+ taken up from the cytosol cause relaxation. The Na+/Ca2+ exchanger (NCX) is an antiporter membrane protein that controls Ca2+ influx and efflux across the membrane. However, the possible relation of NCX in gastric fundus motility is largely unknown. Here, we investigated electric field stimulation (EFS)-induced relaxations in the circular muscles of the gastric fundus in smooth muscle-specific NCX1 transgenic mice (Tg). EFS caused a bi-phasic response, transient and sustained relaxation. The sustained relaxation prolonged for an extended period after the end of the stimulus. EFS-induced transient relaxation and sustained relaxation were greater in Tg than in wild-type mice (WT). Disruption of nitric oxide component by N-nitro-l-arginine, EFS-induced transient and sustained relaxations caused still marked in Tg compared to WT. Inhibition of PACAP by antagonist, EFS-induced sustained relaxation in Tg was not seen, similar to WT. Nevertheless, transient relaxation remained more pronounced in Tg than in WT. Next, we examined responses to NO and PACAP in smooth muscles. The magnitudes of NOR-1, which generates NO, and PACAP-induced relaxations were greater in Tg than in WT. In this study, we demonstrate that NCX1 regulates gastric fundus motility.
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Affiliation(s)
- Yasuyuki Fujimoto
- Laboratory of Veterinary Pharmacology, Division of Veterinary Science, Osaka Prefecture University Graduate School of Life and Environmental Science, Osaka, Japan
| | - Satomi Hayashi
- Laboratory of Veterinary Pharmacology, Division of Veterinary Science, Osaka Prefecture University Graduate School of Life and Environmental Science, Osaka, Japan
| | - Yasu-Taka Azuma
- Laboratory of Veterinary Pharmacology, Division of Veterinary Science, Osaka Prefecture University Graduate School of Life and Environmental Science, Osaka, Japan.
| | - Kazunori Mukai
- Laboratory of Veterinary Pharmacology, Division of Veterinary Science, Osaka Prefecture University Graduate School of Life and Environmental Science, Osaka, Japan
| | - Kazuhiro Nishiyama
- Laboratory of Veterinary Pharmacology, Division of Veterinary Science, Osaka Prefecture University Graduate School of Life and Environmental Science, Osaka, Japan
| | - Satomi Kita
- Department of Pharmacology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan
| | - Ai Morioka
- Laboratory of Veterinary Pharmacology, Division of Veterinary Science, Osaka Prefecture University Graduate School of Life and Environmental Science, Osaka, Japan
| | - Hidemitsu Nakajima
- Laboratory of Veterinary Pharmacology, Division of Veterinary Science, Osaka Prefecture University Graduate School of Life and Environmental Science, Osaka, Japan
| | - Takahiro Iwamoto
- Department of Pharmacology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan
| | - Tadayoshi Takeuchi
- Laboratory of Veterinary Pharmacology, Division of Veterinary Science, Osaka Prefecture University Graduate School of Life and Environmental Science, Osaka, Japan
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Min YW, Hong YS, Ko EJ, Lee JY, Ahn KD, Bae JM, Rhee PL. Nitrergic Pathway Is the Main Contributing Mechanism in the Human Gastric Fundus Relaxation: An In Vitro Study. PLoS One 2016; 11:e0162146. [PMID: 27589594 PMCID: PMC5010257 DOI: 10.1371/journal.pone.0162146] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2016] [Accepted: 08/17/2016] [Indexed: 12/11/2022] Open
Abstract
Background Human gastric fundus relaxation is mediated by intrinsic inhibitory pathway. We investigated the roles of nitrergic and purinergic pathways, two known inhibitory factors in gastric motility, on spontaneous and nerve-evoked contractions in human gastric fundus muscles. Methods Gastric fundus muscle strips (12 circular and 13 longitudinal) were obtained from patients without previous gastrointestinal motility disorder who underwent gastrectomy for stomach cancer. Using these specimens, we examined basal tone, peak, amplitude, and frequency of spontaneous contractions, and peak and nadir values under electrical field stimulation (EFS, 150 V, 0.3 ms, 10 Hz, 20 s). To examine responses to purinergic and nitrergic inhibition without cholinergic innervation, atropine (muscarinic antagonist, 1 μM), MRS2500 (a purinergic P2Y1 receptor antagonist, 1 μM), and N-nitro-L-arginine (L-NNA, a nitric oxide synthase inhibitor, 100 μM) were added sequentially for spontaneous and electrically-stimulated contractions. Tetrodotoxin was used to confirm any neuronal involvement. Results In spontaneous contraction, L-NNA increased basal tone and peak in both muscle layers, while amplitude and frequency were unaffected. EFS (up to 10 Hz) uniformly induced initial contraction and subsequent relaxation in a frequency-dependent manner. Atropine abolished initial on-contraction and induced only relaxation during EFS. While MRS2500 showed no additional influence, L-NNA reversed relaxation (p = 0.012 in circular muscle, and p = 0.006 in longitudinal muscle). Tetrodotoxin abolished any EFS-induced motor response. Conclusions The relaxation of human gastric fundus muscle is reduced by nitrergic inhibition. Hence, nitrergic pathway appears to be the main mechanism for the human gastric fundus relaxation.
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Affiliation(s)
- Yang Won Min
- Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
| | - Yun Soo Hong
- Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
| | - Eun-Ju Ko
- Biomedical Research Institute, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
| | - Ji-Yeon Lee
- Biomedical Research Institute, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
| | - Ki Duck Ahn
- Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
| | - Je Moon Bae
- Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
| | - Poong-Lyul Rhee
- Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
- * E-mail:
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Azuma YT, Hayashi S, Nishiyama K, Kita S, Mukai K, Nakajima H, Iwamoto T, Takeuchi T. Na(+) /Ca(2+) exchanger-heterozygote knockout mice display increased relaxation in gastric fundus and accelerated gastric transit in vivo. Neurogastroenterol Motil 2016; 28:827-36. [PMID: 26787195 DOI: 10.1111/nmo.12779] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/21/2015] [Accepted: 12/25/2015] [Indexed: 01/31/2023]
Abstract
BACKGROUND For the contraction and relaxation of gastric smooth muscles to occur, the intracellular Ca(2+) concentration must be increased and decreased, respectively. The Na(+) /Ca(2+) exchanger (NCX) is a plasma membrane transporter that is involved in regulating intracellular Ca(2+) concentrations. METHODS To determine the role of NCX in gastrointestinal tissues, we examined electric field stimulation (EFS)-induced relaxations in the circular muscles of the gastric fundus in NCX1 and NCX2 heterozygote knockout mice (HET). KEY RESULTS The myenteric plexus layers and the longitudinal and circular muscle layers in the gastric fundus of wild-type mice (WT) were strongly immunoreactive to NCX1 and NCX2. EFS induced a transient relaxation that was apparent during the stimulus and a sustained relaxation that persisted after the end of the stimulus. The amplitudes of EFS-induced transient relaxation and sustained relaxation were greater in NCX1 HET and NCX2 HET than in WT. When an inhibitor of nitric oxide synthase was added following the EFS, neither NCX1 HET nor NCX2 HET exhibited transient relaxation, similar to WT. Furthermore, when a PACAP antagonist was added following the EFS, sustained relaxation in NCX1 HET and NCX2 HET was not observed, similar to WT. Next, we examined the effect of NCX heterozygous deficiency on relaxation in response to NO and PACAP in smooth muscles. The magnitude of NOR-1- and PACAP-induced relaxations in NCX1 HET and NCX2 HET was similar to that of WT. CONCLUSIONS & INFERENCES In this study, we demonstrate that NCX1 and NCX2 expressed in neurons regulate the motility in the gastric fundus.
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Affiliation(s)
- Y T Azuma
- Laboratory of Veterinary Pharmacology, Division of Veterinary Science, Osaka Prefecture University Graduate School of Life and Environmental Science, Osaka, Japan
| | - S Hayashi
- Laboratory of Veterinary Pharmacology, Division of Veterinary Science, Osaka Prefecture University Graduate School of Life and Environmental Science, Osaka, Japan
| | - K Nishiyama
- Laboratory of Veterinary Pharmacology, Division of Veterinary Science, Osaka Prefecture University Graduate School of Life and Environmental Science, Osaka, Japan
| | - S Kita
- Department of Pharmacology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan
| | - K Mukai
- Laboratory of Veterinary Pharmacology, Division of Veterinary Science, Osaka Prefecture University Graduate School of Life and Environmental Science, Osaka, Japan
| | - H Nakajima
- Laboratory of Veterinary Pharmacology, Division of Veterinary Science, Osaka Prefecture University Graduate School of Life and Environmental Science, Osaka, Japan
| | - T Iwamoto
- Department of Pharmacology, Faculty of Medicine, Fukuoka University, Fukuoka, Japan
| | - T Takeuchi
- Laboratory of Veterinary Pharmacology, Division of Veterinary Science, Osaka Prefecture University Graduate School of Life and Environmental Science, Osaka, Japan
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Squecco R, Garella R, Francini F, Baccari MC. Influence of obestatin on the gastric longitudinal smooth muscle from mice: mechanical and electrophysiological studies. Am J Physiol Gastrointest Liver Physiol 2013; 305:G628-37. [PMID: 23989009 DOI: 10.1152/ajpgi.00059.2013] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Obestatin is a hormone released from the stomach deriving from the same peptide precursor as ghrelin. It is known to act as an anorectic hormone decreasing food intake, but contrasting results have been reported about the effects of obestatin on gastrointestinal motility. The aim of the present study was to investigate whether this peptide may act on the gastric longitudinal smooth muscle by using a combined mechanical and electrophysiological approach. When fundal strips from mice were mounted in organ baths for isometric recording of the mechanical activity, obestatin caused a tetrodotoxin-insensitive decrease of the basal tension and a reduction in amplitude of the neurally induced cholinergic contractile responses, even in the presence of the nitric oxide synthesis inhibitor N(G)-nitro-l-arginine. Obestatin reduced the amplitude of the response to the ganglionic stimulating agent dimethylphenyl piperazinium iodide but did not influence that to methacholine. In nonadrenergic, noncholinergic conditions, obestatin still decreased the basal tension of the preparations without influencing the neurally induced relaxant responses. For comparison, in circular fundal strips, obestatin had no effects. Notably, in the longitudinal antral ones, obestatin only caused a decrease of the basal tension. Electrophysiological experiments, performed by a single microelectrode inserted in a gastric longitudinal smooth muscle cell, showed that obestatin had similar effects in fundal and antral preparations: it decreased the resting specific membrane conductance, inhibited Ca(2+) currents, and positively shifted their voltage threshold of activation. In conclusion, the present results indicate that obestatin influences gastric smooth muscle exerting site-specific effects.
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Affiliation(s)
- Roberta Squecco
- Dipartimento di Medicina Sperimentale e Clinica, Sezione di Scienze Fisiologiche, Università degli Studi, Firenze, Viale Morgagni 63, 50134, Italy.
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Garella R, Baccari MC. Endocannabinoids modulate non-adrenergic, non-cholinergic inhibitory neurotransmission in strips from the mouse gastric fundus. Acta Physiol (Oxf) 2012; 206:80-7. [PMID: 22510304 DOI: 10.1111/j.1748-1716.2012.02444.x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2011] [Revised: 12/06/2011] [Accepted: 04/10/2012] [Indexed: 02/06/2023]
Abstract
AIM To investigate the effects of endocannabinoids on non-adrenergic, non-cholinergic (NANC) relaxant responses in gastric strips from mice. METHODS Gastric longitudinal strips from the fundus region were mounted in organ baths for isometric recording. RESULTS In carbachol-precontracted strips, electrical field stimulation (EFS) elicited tetrodotoxin (TTX)-sensitive fast nitrergic relaxant responses that were followed, at the highest stimulation frequency, by sustained relaxations. The latter were abolished by α-chymotrypsin. Anandamide caused a TTX-sensitive relaxation that was abolished by α-chymotrypsin but unaffected by the nitric oxide (NO) synthesis inhibitor, Nω-nitro-L-arginine (L-NNA). Anandamide reduced the amplitude of EFS-induced fast relaxations, whereas increased that of sustained ones. Relaxation to the nicotinic receptor agonist dimethylphenyl piperazinium iodide (DMPP) was decreased in amplitude by either anandamide or L-NNA, whereas, surprisingly, it was increased by α-chymotrypsin and abolished by L-NNA plus α-chymotrypsin. Relaxation to vasoactive intestinal polypeptide (VIP) was not influenced by anandamide or L-NNA and was abolished by α-chymotrypsin. Following VIP desensitization, fast relaxant responses to EFS were reduced and the sustained ones abolished. The CB1 receptor antagonist AM251 increased, only at the highest stimulation frequency, the amplitude of the EFS-induced fast relaxation and reduced the sustained one. AM251 increased the response to DMPP and abolished that to anandamide. The CB2 receptor antagonist AM630 had no effects. CONCLUSION These results indicate that endocannabinoids modulate, via prejunctional CB1 receptors, the NANC peptidergic neurotransmission that, in turn, affects the nitrergic one.
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Affiliation(s)
- R. Garella
- Department of Physiological Sciences; University of Florence; Florence; Italy
| | - M. C. Baccari
- Department of Physiological Sciences; University of Florence; Florence; Italy
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Garella R, Baccari MC. Contribution of endogenous nitrergic and peptidergic influences to the altered neurally-induced gastric contractile responses in strips from dystrophic (mdx) mice. ACTA ACUST UNITED AC 2009; 160:57-63. [PMID: 20035804 DOI: 10.1016/j.regpep.2009.12.012] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2009] [Revised: 11/27/2009] [Accepted: 12/17/2009] [Indexed: 02/08/2023]
Abstract
Gastrointestinal motor disorders have been reported to occur in dystrophic (mdx) mice. The aim of the present study was to investigate the contribution of endogenous nitrergic and peptidergic components to the gastric contractile responses in strips from wild type (WT) and mdx mice. In both preparations, electrical field stimulation (EFS) induced frequency-dependent excitatory responses that were abolished by atropine or tetrodotoxin. The amplitude of the neurally-induced contractile responses was greater in strips from mdx mice in respect to the WT ones. In both preparations, at the end of the stimulation period strip tension decayed below the pre-stimulus level (off-relaxations). The nitric oxide (NO) synthesis inhibitor L-NNA increased the amplitude of the EFS-induced contractile responses without influencing off-relaxations. alpha-chymotrypsin and PACAP 6-38 abolished off-relaxations and also caused a reduction in amplitude of the contractile responses, whereas VIP receptor antagonists were ineffective. The efficacy of L-NNA, alpha-chymotrypsin or PACAP 6-38 on the excitatory responses was lower in strips from mdx mice in respect to the WT ones. alpha-chymotrypsin, in the presence of L-NNA, was no longer able to decrease the amplitude of the neurally-induced contractile responses but still abolished off-relaxations in both preparations. Direct muscular responses to methacholine were similar in amplitude in the two preparations and were not influenced by L-NNA or alpha-chymotrypsin. The results indicate that both endogenous NO and peptides influence the EFS-induced cholinergic responses: a stronger peptidergic modulatory action on a weaker nitrergic neurotransmission is suggested to occur in strips from mdx mice in respect to the WT ones and to contribute to the altered gastric contractile responses.
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Affiliation(s)
- Rachele Garella
- Department of Physiological Sciences, University of Florence, I-50134 Florence, Italy
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Baccari MC, Bani D, Calamai F. Evidence for a modulatory role of orexin A on the nitrergic neurotransmission in the mouse gastric fundus. ACTA ACUST UNITED AC 2008; 154:54-9. [PMID: 19150469 DOI: 10.1016/j.regpep.2008.12.005] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2008] [Revised: 11/12/2008] [Accepted: 12/15/2008] [Indexed: 12/12/2022]
Abstract
The presence of orexins and their receptors in the gastrointestinal tract supports a local action of these peptides. Aim of the present study was to investigate the effects of orexin A (OXA) on the relaxant responses of the mouse gastric fundus. Mechanical responses of gastric strips were recorded via force-displacement transducers. The presence of orexin receptors (OX-1R) was also evaluated by immunocytochemistry. In carbachol precontracted strips and in the presence of guanethidine, electrical field stimulation (EFS) elicited a fast inhibitory response that may be followed, at the highest stimulation frequencies employed, by a sustained relaxation. All relaxant responses were abolished by TTX. The fast response was abolished by the nitric oxide (NO) synthesis inhibitor l-NNA (2x10(-4) M) as well as by the guanylate cyclase inhibitor ODQ (1x10(-6) M). OXA (3x10(-7) M) greatly increased the amplitude of the EFS-induced fast relaxation without affecting the sustained one. OXA also potentiated the amplitude of the relaxant responses elicited by the ganglionic stimulating agent DMPP (1x10(-5) M), but had no effects on the direct smooth muscle relaxant responses elicited by papaverine (1x10(-5) M) or VIP (1x10(-7) M). In the presence of l-NNA, the response to DMPP was reduced in amplitude and no longer influenced by OXA. The OX1 receptor antagonist SB-334867 (1x10(-5) M) reduced the amplitude of the EFS-induced fast relaxation without influencing neither the sustained responses nor those to papaverine and VIP. Immunocytochemistry showed the presence of neurons that co-express neuronal nitric oxide synthase and OX-1R. These results indicate that, in mouse gastric fundus, OXA exerts a modulatory action at the postganglionic level on the nitrergic neurotransmission.
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Hagi K, Azuma YT, Nakajima H, Shintani N, Hashimoto H, Baba A, Takeuchi T. Involvements of PHI-nitric oxide and PACAP-BK channel in the sustained relaxation of mouse gastric fundus. Eur J Pharmacol 2008; 590:80-6. [PMID: 18602629 DOI: 10.1016/j.ejphar.2008.05.045] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2007] [Revised: 05/01/2008] [Accepted: 05/20/2008] [Indexed: 12/16/2022]
Abstract
The roles of nitric oxide (NO) and K(+) channels in sustained relaxation induced by electrical field stimulation (EFS) in the presence of atropine and guanethidine were studied in circular muscle strips of mouse gastric fundus. In the wild-type mouse, N(G)-nitro-l-arginine (l-nitroarginine), a nitric oxide synthase inhibitor, significantly inhibited the sustained relaxation in addition to the rapid relaxation. The sustained relaxation in pituitary adenylate cyclase-activating peptide (PACAP)-knockout mouse, which was smaller than that of the wild-type mouse, was also inhibited by l-nitroarginine. l-Nitroarginine inhibited the relaxation induced by the peptide histidine isoleucine (PHI), but not that induced by PACAP. S-Nitroso-N-acetyl-dl-penicillamine (SNAP), a NO donor, -induced relaxation was not affected by PACAP(6-38). EFS-induced sustained relaxation was inhibited by iberiotoxin, a big conductance calcium-activated K(+) (BK) channel inhibitor, but not by apamin, a small conductance calcium-activated K(+) (SK) channel inhibitor, and glibenclamide, an ATP-sensitive K(+) channel inhibitor. The relaxation that remained after the iberiotoxin-treatment was significantly inhibited by l-nitroarginine. Iberiotoxin inhibited PACAP-induced relaxation, while it had no effect on both PHI- and SNAP-induced relaxation. Immunoreactivities to anti-BK channel and anti-PHI antibodies were found in the circular muscle and the myenteric plexus layers, respectively. These results suggest interplay between PHI and NO in the sustained relaxation of the mouse gastric fundus, and that BK channels are involved in the PACAP-component of the sustained relaxation.
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Affiliation(s)
- Kiyomi Hagi
- Laboratory of Veterinary Pharmacology, Graduate School of Life and Environmental Science, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai 599-8531, Japan
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Regulation of basal tone, relaxation and contraction of the lower oesophageal sphincter. Relevance to drug discovery for oesophageal disorders. Br J Pharmacol 2007; 153:858-69. [PMID: 17994108 DOI: 10.1038/sj.bjp.0707572] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
The lower oesophageal sphincter (LOS) is a specialized region of the oesophageal circular smooth muscle that allows the passage of a swallowed bolus to the stomach and prevents the reflux of gastric contents into the oesophagus. The anatomical arrangement of the LOS includes semicircular clasp fibres adjacent to the lesser gastric curvature and sling fibres following the greater gastric curvature. Such anatomical arrangement together with an asymmetric intrinsic innervation and distinct proportion of neurotransmitters in both regions produces an asymmetric pressure profile. The LOS tone is myogenic in origin and depends on smooth muscle properties that lead to opening of L-type Ca(2+) channels; however it can be modulated by enteric motor neurons, the parasympathetic and sympathetic extrinsic nervous system and several neurohumoral substances. Nitric oxide synthesized by neuronal NOS is the main inhibitory neurotransmitter involved in LOS relaxation. Different putative neurotransmitters have been proposed to play a role together with NO. So far, only ATP or related purines have shown to be co-transmitters with NO. Acetylcholine and tachykinins are involved in the LOS contraction acting through acetylcholine M(3) and tachykinin NK(2) receptors. Nitric oxide can also be involved in the regulation of LOS contraction. The understanding of the mechanisms that originate and modulate LOS tone, relaxation and contraction and the characterization of neurotransmitters and receptors involved in LOS function are important to develop new pharmacological tools to treat primary oesophageal motor disorders and gastro-oesophageal reflux disease.
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Baccari MC, Calamai F. Influence of orexin A on the mechanical activity of mouse gastric strips. ACTA ACUST UNITED AC 2007; 146:67-72. [PMID: 17881068 DOI: 10.1016/j.regpep.2007.08.004] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2007] [Revised: 07/16/2007] [Accepted: 08/09/2007] [Indexed: 10/22/2022]
Abstract
The presence of orexins and orexin receptors has been revealed not only in the central nervous system but also in the gastrointestinal tract. The present study was aimed to investigate the influence of orexin A (OXA) on the mechanical activity of fundal and antral strips of the mouse stomach. In the fundus, electrical field stimulation (EFS) elicited tetrodotoxin (TTX)-sensitive, frequency-dependent contractile responses whose amplitude was markedly reduced by OXA and enhanced by the orexin-1 type receptor antagonist SB-334867. In the presence of the NO synthesis inhibitor L-N(G)-nitro arginine (L-NNA), OXA was no longer effective. Methacholine caused a sustained contracture whose amplitude was not influenced by OXA, TTX or L-NNA. In carbachol-precontracted strips, the neurally-induced relaxant responses elicited during EFS were increased in amplitude by OXA. Antral strips showed a spontaneous contractile activity that was unaffected by TTX or L-NNA and transiently depressed by EFS. OXA did not influence either the spontaneous motility or the EFS-induced effects. The results indicate that OXA exerts region-specific effects and that, in the fundus, depresses EFS-induced contractile responses by acting at the nervous level. It is likely that NO is involved in the effects of the peptide.
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Affiliation(s)
- Maria Caterina Baccari
- Department of Physiological Sciences, University of Florence, Viale G.B. Morgagni 63, 50134, Florence, Italy.
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Takeuchi T, Toyoshima M, Mukai K, Hagi K, Matsui M, Nakajima H, Azuma YT, Hata F. Involvement of M(2) muscarinic receptors in relaxant response of circular muscle of mouse gastric antrum. Neurogastroenterol Motil 2006; 18:226-33. [PMID: 16487414 DOI: 10.1111/j.1365-2982.2005.00733.x] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Our previous study showed that atropine significantly inhibited the sustained relaxation induced by electrical field stimulation (EFS) in the circular muscle strips prepared from the mouse antrum, and that pituitary adenylate cyclase activating peptide (PACAP) partially mediated the sustained relaxation. The muscarinic receptor subtype associated with the sustained relaxation was examined in the present study by using each muscarinic receptor subtype of knockout (KO) mice. EFS-induced sustained relaxation in the antrum prepared from M(2) receptor KO mice was significantly less than that of wild-type mice. Atropine failed to inhibit this relaxation. On the other hand, similar sustained relaxation and inhibitory effects of atropine to those of wild-type mice were observed in M(1), M(3) and M(4) receptor KO mice. Exogenously added PACAP-27 relaxed the antral strips of wild-type and M(2) receptor KO mice to a similar extent. Immunohistochemical study revealed that M(2) receptor immunoreactivity was localized with PACAP-immunoreactivity in enteric neurons within the antrum of wild-type mice. In contrast, atropine did not affect the EFS-induced sustained relaxation in the gastric fundus. These results suggest that M(2) receptors modulate the sustained relaxation, probably through the regulation of PACAP release, in the mouse antrum.
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Affiliation(s)
- T Takeuchi
- Department of Veterinary Pharmacology, Graduate School of Agriculture and Life Science, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai 599-8531, Japan.
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Toyoshima M, Takeuchi T, Goto H, Mukai K, Shintani N, Hashimoto H, Baba A, Hata F. Roles of PACAP and PHI as inhibitory neurotransmitters in the circular muscle of mouse antrum. Pflugers Arch 2005; 451:559-68. [PMID: 16292577 DOI: 10.1007/s00424-005-1491-6] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2005] [Accepted: 07/04/2005] [Indexed: 11/24/2022]
Abstract
Mediators of neurogenic responses of the gastric antrum were studied in wild-type and pituitary adenylate cyclase-activating polypeptide (PACAP) -knockout (KO) mice. Electrical field stimulation (EFS) to the circular muscle strips of the wild-type mouse antrum induced a triphasic response; rapid transient relaxation and contraction, and sustained relaxation that was prolonged for an extended period after the end of EFS. The transient relaxation and contraction were completely inhibited by L-nitroarginine and atropine, respectively. The sustained relaxation was significantly inhibited by a PACAP receptor antagonist, PACAP(6-38). The antral strips prepared from PACAP-KO mice unexpectedly exhibited a tri-phasic response. However, the sustained relaxation was decreased to about one-half of that observed in wild-type mice. PACAP(6-38) inhibited EFS-induced sustained relaxation (33.5% of control) in PACAP-KO mice. Anti-peptide histidine isoleucine (PHI) serum partially (the 30% inhibition) or significantly (the 60% inhibition) inhibited the sustained relaxations in the wild-type and PACAP-KO mice, respectively. The immunoreactivities to the anti-PACAP and anti-PHI serums were found in myenteric ganglia of the mouse antrum. These results suggest that nitric oxide and acetylcholine mediate the transient relaxation and contraction, respectively, and that PACAP and PHI separately mediate the sustained relaxation in the antrum of the mouse stomach.
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Affiliation(s)
- Makiko Toyoshima
- Department of Veterinary Pharmacology, Graduate School of Agriculture and Life Science, Osaka Prefecture University, Sakai, Japan
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Mukai K, Takeuchi T, Toyoshima M, Satoh Y, Fujita A, Shintani N, Hashimoto H, Baba A, Hata F. PACAP- and PHI-mediated sustained relaxation in circular muscle of gastric fundus: findings obtained in PACAP knockout mice. ACTA ACUST UNITED AC 2005; 133:54-61. [PMID: 16229904 DOI: 10.1016/j.regpep.2005.09.019] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2005] [Revised: 09/09/2005] [Accepted: 09/12/2005] [Indexed: 12/31/2022]
Abstract
Mediators of neurogenic responses of the gastric fundus were studied in wild type and pituitary adenylate cyclase activating peptide (PACAP) knockout mice. Electrical field stimulation (EFS) to the circular muscle strips of the wild type mouse fundus induced a tri-phasic response, rapid transient contraction and relaxation, and sustained relaxation that was prolonged for an extended period after the end of EFS. The transient relaxation and contraction were completely inhibited by N(G)-nitro-L-arginine and atropine, respectively. The sustained relaxation was completely inhibited by a PACAP receptors antagonist, PACAP(6-38). The strips prepared from PACAP knockout mice exhibited a large contraction without rapid relaxation and unexpectedly, a sustained relaxation. However, the sustained relaxation was decreased to about a half of that observed in wild type mice. Anti-peptide histidine isoleucine (PHI) serum abolished the sustained relaxation in the knockout mice. The serum partially inhibited the sustained relaxation in wild type mice and PACAP(6-38) abolished the relaxation that remained after the antiserum-treatment. PHI relaxed the strips prepared from wild type mice. The relaxation was completely inhibited by PACAP(6-38). It was concluded that PACAP and PHI separately mediate the sustained relaxation in the mouse gastric fundus, and that nitric oxide and ACh mediate transient relaxation and contraction, respectively.
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Affiliation(s)
- Kazunori Mukai
- Department of Veterinary Pharmacology, Graduate School of Agriculture and Life Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan
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Baccari MC, Bani D, Bigazzi M, Calamai F. Influence of relaxin on the neurally induced relaxant responses of the mouse gastric fundus. Biol Reprod 2004; 71:1325-9. [PMID: 15215200 DOI: 10.1095/biolreprod.104.029579] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
The peptide hormone relaxin has been reported to depress the amplitude of contractile responses in the mouse gastric fundus by upregulating nitric oxide (NO) biosynthesis at the neural level. In the present study, we investigated whether relaxin also influenced nonadrenergic, noncholinergic (NANC) gastric relaxant responses in mice. Female mice in proestrus or estrus were treated for 18 h with relaxin (1 microg s.c.) or vehicle (controls). Mechanical responses of gastric fundal strips were recorded via force-displacement transducers. In carbachol precontracted strips from control mice and in the presence of guanethidine, electrical field stimulation (EFS) elicited fast relaxant responses that may be followed by a sustained relaxation. All relaxant responses were abolished by tetrodotoxin. Relaxin increased the amplitude of the EFS-induced fast relaxation without affecting either the sustained one or the direct smooth muscle response to papaverine. In the presence of the NO synthesis inhibitor L-N(G)-nitro arginine (L-NNA), that abolished the EFS-induced fast relaxation without influencing the sustained one, relaxin was ineffective. In strips from relaxin-pretreated mice, EFS-induced fast relaxations were enhanced in amplitude with respect to the controls, while sustained ones as well as direct smooth muscle responses to papaverine were not changed. Further addition of relaxin to the bath medium did not influence neurally induced fast relaxant responses, whereas L-NNA did. In conclusion, in the mouse gastric fundus, relaxin enhances the neurally induced nitrergic relaxant responses acting at the neural level.
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Baccari MC, Nistri S, Quattrone S, Bigazzi M, Bani Sacchi T, Calamai F, Bani D. Depression by relaxin of neurally induced contractile responses in the mouse gastric fundus. Biol Reprod 2003; 70:222-8. [PMID: 14522837 DOI: 10.1095/biolreprod.103.018374] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023] Open
Abstract
The peptide hormone relaxin, which attains high circulating levels during pregnancy, has been shown to depress small-bowel motility through a nitric oxide (NO)-mediated mechanism. In the present study we investigated whether relaxin also influences gastric contractile responses in mice. Female mice in proestrus or estrus were treated for 18 h with relaxin (1 microg s.c.) or vehicle (controls). Mechanical responses of gastric fundal strips were recorded via force-displacement transducers. Evaluation of the expression of nitric oxide synthase (NOS) isoforms was performed by immunohistochemistry and Western blot. In control mice, neurally induced contractile responses elicited by electrical field stimulation (EFS) were reduced in amplitude by addition of relaxin to the organ bath medium. In the presence of the NO synthesis inhibitor l-NNA, relaxin was ineffective. Direct smooth muscle contractile responses were not influenced by relaxin or l-NNA. In strips from relaxin-pretreated mice, the amplitude of neurally induced contractile responses was also reduced in respect to the controls, while that of direct smooth muscle contractions was not. Further addition of relaxin to the bath medium did not influence EFS-induced responses, whereas l-NNA did. An increased expression of NOS I and NOS III was observed in gastric tissues from relaxin-pretreated mice. In conclusion, the peptide hormone relaxin depresses cholinergic contractile responses in the mouse gastric fundus by up-regulating NO biosynthesis at the neural level.
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Affiliation(s)
- Maria Caterina Baccari
- Departments of Physiological Sciences and Anatomy,4, University of Florence, Florence, Italy.
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Stengel PW, Cohen ML. M1 receptor-mediated nitric oxide-dependent relaxation unmasked in stomach fundus from M3 receptor knockout mice. J Pharmacol Exp Ther 2003; 304:675-82. [PMID: 12538821 DOI: 10.1124/jpet.102.042283] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Muscarinic receptors can mediate both contractile and relaxant responses in smooth muscle. The stomach fundus from wild-type mice possesses a neuronal M(1) receptor that mediates relaxation to carbamylcholine and (4-hydroxy-2-butynyl)-1-trimethylammonium-3-chlorocarbanilate chloride (McN-A-343) but is masked by M(3) receptor-mediated contraction to both agonists. When the M(3) receptor was deleted, cholinergic-induced relaxation was unmasked. M(1) receptor antagonism with pirenzepine, nitric oxide (NO) synthase inhibition with N(omega)-nitro-L-arginine methyl ester hydrochloride, and inhibition of neuronal activation with tetrodotoxin abolished relaxation to McN-A-343 in tissues from M(3) receptor knockout mice, supporting the neuronal localization of an M(1) receptor that activated NO release to effect relaxation. However, the cyclooxygenase inhibitor indomethacin did not affect contraction or relaxation to carbamylcholine in stomach fundus from wild-type or M(3) receptor knockout mice, indicating that cyclooxygenase products played no role in these responses. The neuronal M(1) receptor modulated relaxation induced by carbamylcholine and McN-A-343 but not relaxation induced by electric field stimulation of the stomach fundus. These data support the presence of M(1) receptor-mediated relaxation in the stomach and suggest that when the M(3) receptor is eliminated or blocked, M(1) receptor-mediated gastric relaxation may be enhanced, possibly leading to alterations in gastric emptying and subsequent effects on body weight.
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Affiliation(s)
- Peter W Stengel
- Eli Lilly and Company, Lilly Research Laboratories, Neuroscience Research, Lilly Corporate Center, Indianapolis, Indiana 46285, USA.
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