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Vasomotion is the oscillation of vascular tone which gives rise to flow motion of blood into an organ. As is well known, spontaneous contractile organs such as heart, GI, and genitourinary tract produce rhythmic contraction. It imposes or removes pressure on their vessels alternatively for exchange of many substances. It was first described over 150 years ago, however the physiological mechanism and pathophysiological implications are not well understood. This study aimed to elucidate underlying mechanisms and physiological function of vasomotion in human arteries. Conventional contractile force measurement, immunohistochemistry, and Western blot analysis were employed to study human left gastric artery (HLGA) and uterine arteries (HUA). RESULTS: Circular muscle of HLGA and/or HUA produced sustained tonic contraction by high K+ (50 mM) which was blocked by 2 µM nifedipine. Stepwise stretch and high K+ produced nerve-independent spontaneous contraction (vasomotion) (around 45% of tested tissues). Vasomotion was also produced by application of BayK 8644, 5-HT, prostagrandins, oxytocin. It was blocked by nifedipine (2 µM) and blockers of intracellular Ca2+ stores. Inhibitors of Ca2+ -activated Cl- channels (DIDS and/or niflumic acid) and ATP-sensitive K+ (KATP ) channels inhibited vasomotion reversibly. Metabolic inhibition by sodium cyanide (NaCN) and several neuropeptides also regulated vasomotion in KATP channel-sensitive and -insensitive manner. Finally, we identified TMEM16A Ca2+ -activated Cl- channels and subunits of KATP channels (Kir 6.1/6.2 and sulfonylurea receptor 2B [SUR2B]), and c-Kit positivity by Western blot analysis. We conclude that vasomotion is sensitive to TMEM16A Ca2+ -activated Cl- channels and metabolic changes in human gastric and uterine arteries. Vasomotion might play an important role in the regulation of microcirculation dynamics even in pacemaker-related autonomic contractile organs in humans.
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Artérias , Canais Iônicos , Contração Isométrica , Humanos , Canais Iônicos/fisiologia , Nifedipino/farmacologia , Artéria Uterina , Artérias/fisiologiaRESUMO
Background/Aims: The gastrointestinal symptom of diabetes mellitus, chronic constipation, seriously affects patients' life. Whereas, the mechanism of chronic constipation is still ambiguous, resulting in a lack of effective therapies for this symptom. As a part of the smooth muscle cells, interstitial cells of Cajal, and platelet-derived growth factor receptor alpha-positive (PDGFRα+) cells syncytium (SIP syncytium), PDGFRα+ cells play an important role in regulating colonic motility. According to our previous study, in PDGFRα+ cells in colons of diabetic mice, the function of the P2Y1 purinergic receptor/type 3 small-conductance calcium-activated potassium (SK3) channel signaling pathway is strengthened, which may lead to colonic dysmotility. The purpose of this study is to investigate the changes in SK3 channel properties of PDGFRα+ cells in diabetic mice. Methods: Whole-cell patch clamp, Western blotting, superoxide dismutase activity measurement, and malondialdehyde measurement were main methods in the present study. Results: The present study revealed that when dialysed with low calcium ion (Ca2+) solution, the SK3 current density was significantly decreased in PDGFRα+ cells from diabetic mice. However, the SK3 current density in PDGFRα+ cells was enhanced from diabetic mice when dialysed with high Ca2+ solution. Moreover, hydrogen peroxide-treatment mimicked this phenomenon in SK3 transgenic HEK293 cells. The subunit of SK3 channels, protein kinase CK2, was up-regulated in colonic muscle layers and hydrogen peroxide-treated HEK293 cells. Additionally, protein phosphatase 2A, the subunit of SK3 channels, was not changed in streptozotocin-treated mouse colons or hydrogen peroxide-treated HEK293 cells. Conclusion: The diabetic oxidative stress-induced upregulation of CK2 contributed to modulating SK3 channel sensitivity to Ca2+ in colonic PDGFRα+ cells, which may result in colonic dysmotility in diabetic mice.
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BACKGROUND: Peripheral corticotropin-releasing factor (CRF) has been reported to affect gastrointestinal motility through corticotropin-releasing factor receptor located in enteric nervous system (ENS), but less is known about of the relationship between peripheral CRF and interstitial cells of Cajal (ICC). METHODS: Mice were intraperitoneally injected with CRF receptor agonists to determine their effects on colonic ICC. Chronic heterotypic stress (CHeS) was applied to mice to determine endogenous CRF-CRF receptor signaling on colonic ICC. RESULTS: We found that stressin1, a selective CRF receptor 1 (CRF1 ) agonist, significantly increased the expression of CRF1 but had no effect on the expression of CRF2 in the smooth muscles of murine colon. The protein expression of c-Kit, Anoctamin-1 (ANO1), and stem cell factor (SCF) in the colonic smooth muscles was significantly decreased in stressin1-treated mice. Accordingly, 2-(4-Chloro-2-methylphenoxy)-N'-(2-methoxybenzylidene) acetohydrazide (Ani 9), a selective ANO1 blocker, had a less significant inhibitory effect on CMMC in stressin1-treated mice compared to the saline-treated ones. Similarly, we also found that ICC and ANO1 were reduced in the colonic smooth muscles of mice by treatment with sauvagine (ip), a CRF2 agonist. However, different with stressin1, sauvagine decreased the expression of CRF2 besides increasing CRF1 expression in the colonic smooth muscles. Similar results of CRF1 and c-Kit expressions were also obtained from the colon of CHeS-treated mice. CONCLUSION: All these results suggest that CRF may be involved in the abnormality of colonic motility through peripheral CRF1 to decrease the number and function of ICC, which provides a potential target for treating stress-induced gastrointestinal motility disorder.
Assuntos
Células Intersticiais de Cajal , Receptores de Hormônio Liberador da Corticotropina , Camundongos , Animais , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Hormônio Liberador da Corticotropina/farmacologia , Células Intersticiais de Cajal/metabolismo , Colo/metabolismoRESUMO
Our previous study indicated that streptozotocin (STZ)-induced diabetes leads to colonic platelet-derived growth factor receptor-α-positive (PDGFRα+ ) cell proliferation accompanied by slow colonic transit in mice; however, the mechanism of this effect is unclear. The present study used western blotting, immunohistochemistry, and quantitative PCR to investigate whether proteinase-activated receptor 2 (PAR2) mediates PDGFRα+ cell proliferation. Our results showed that PDGFRα, PAR2, and Ki-67 coexpression was increased in the diabetic colonic muscle layer. PDGFRα and PAR2 mRNA and protein expression levels were also markedly enhanced in the diabetic colonic muscle layer. Mice treated with 2-furoyl-LIGRLO-amide (2-F-L-a), a PAR2 agonist, exhibited significant colon elongation and increased smooth muscle weight. In the 2-F-L-a-treated mice, PDGFRα, PAR2, and Ki-67 coexpression was increased and PDGFRα and PAR2 mRNA and protein expression was significantly enhanced in the colonic smooth muscle layer. 2-F-L-a also increased proliferation and PDGFRα expression in NIH/3T3 cells cultured in high glucose, while LY294002, a PI3K antagonist, decreased cell proliferation and PDGFRα expression. PI3K and Akt protein and mRNA expression and p-Akt protein expression in diabetic and 2-F-L-a-treated mice were markedly reduced in colonic smooth muscle. 2-F-L-a also reduced PI3K, Akt, and p-Akt protein expression in NIH/3T3 cells, while the PI3K antagonist LY294002 increased this expression. The results indicate that PAR2 is involved in the proliferation of PDGFRα+ cells through the PI3K/Akt signaling pathway in the colon of STZ-induced diabetic mice, which may contribute to the slow transit and constipation that are associated with diabetes.
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Proliferação de Células , Colo/metabolismo , Diabetes Mellitus Experimental/metabolismo , Receptor PAR-2/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Células Cultivadas , Colo/citologia , Colo/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/fisiologia , Células NIH 3T3 , Oligopeptídeos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor PAR-2/agonistas , Receptor PAR-2/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Transdução de SinaisRESUMO
Under physiological conditions, the motility of smooth muscle in digestive tract is mainly regulated by enteric nervous system (ENS). However, how neural signal is transmitted to smooth muscle is not fully understood. Autonomic nerve endings in the smooth muscle layer form large number of varicosities which contain neurotransmitters. It was considered that nerve pulses arriving at the varicosities may cause the release of neurotransmitters, which may diffuse to the smooth muscle cells to induce contractile or relaxant responses. Over the past decade, a new understanding of the neurotransmission between ENS and smooth muscle has emerged, which emphasizes the role of a functional syncytium consisting of the interstitial cells of Cajal (ICC), the platelet-derived growth factor receptor α positive (PDGFRα+) cells and the smooth muscle cells. Within the syncytium, purine neurotransmitters bind to P2Y1 receptors on PDGFRα+ cells, activating small-conductance calcium activated potassium channel (SK3) to hyperpolarize PDGFRα+ cells, and thus hyperpolarize smooth muscle cells through gap junction, resulting in relaxation of smooth muscle. In this paper, we review the research progress in the field of inhibitory purinergic neurotransmission in the gastrointestinal tract.
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Células Intersticiais de Cajal , Músculo Liso , Miócitos de Músculo Liso , Receptor alfa de Fator de Crescimento Derivado de Plaquetas , Transmissão SinápticaRESUMO
Gastric motility is controlled by slow waves. In general, the activation of the ATP-sensitive K+ (KATP) channels in the smooth muscle opposes the membrane excitability and produces relaxation. Since metabolic inhibition and/or diabetes mellitus are accompanied by dysfunctions of gastric smooth muscle, we examined the possible roles of KATP channels in human gastric motility. We used human gastric corpus and antrum smooth muscle preparations and recorded the mechanical activities with a conventional contractile measuring system. We also identified the subunits of the KATP channels using Western blot. Pinacidil (10 µM), a KATP channel opener, suppressed contractions to 30% (basal tone to -0.2â g) of the control. The inhibitory effect of pinacidil on contraction was reversed to 59% of the control by glibenclamide (20 µM), a KATP channel blocker. The relaxation by pinacidil was not affected by a pretreatment with L-arginine methyl ester, tetraethylammonium, or 4-aminopyridine. Pinacidil also inhibited the acetylcholine (ACh)-induced tonic and phasic contractions in a glibenclamide-sensitive manner (42% and 6% of the control, respectively). Other KATP channel openers such as diazoxide, cromakalim and nicorandil also inhibited the spontaneous and ACh-induced contractions. Calcitonin gene-related peptide (CGRP), a gastric neuropeptide, induced muscle relaxation by the activation of KATP channels in human gastric smooth muscle. Finally, we have found with Western blot studies, that human gastric smooth muscle expressed KATP channels which were composed of Kir 6.2 and SUR2B subunits.
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Canais KATP/metabolismo , Canais KATP/fisiologia , Músculo Liso/fisiologia , Estômago/fisiologia , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Motilidade Gastrointestinal/efeitos dos fármacos , Glibureto/farmacologia , Humanos , Técnicas In Vitro , Canais KATP/antagonistas & inibidores , Contração Muscular/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/químicaRESUMO
As a naturally occurring flavone, luteolin has received much attention due to its antioxidant, anti-inflammatory and anticancer functions. In the present study, we investigated the effect of luteolin on colonic motility and its mechanism using isometric muscle recording and the whole-cell patch-clamp technique in mice. Luteolin dose-dependently inhibited colonic smooth muscles motility and CMMC significantly. BayK8644, an L-type Ca2+ channel agonist, significantly attenuated the luteolin-induced inhibition. Moreover, the calcium currents recorded in colonic smooth muscle cells were dramatically inhibited by luteolin. However, no significant changes were found in the luteolin-induced inhibitory effect in the presence of TEA, a nonselective K+ channel blocker, glibenclamide, an ATP-dependent K+ channel blocker, and apamin, a small-conductance Ca2+-activated K+ channel blocker. Additionally, luteolin did not affect potassium currents. Furthermore, TTX, a Na+ channel blocker, L-NAME, an inhibitor of nitric oxide (NO) synthase, ODQ, an inhibitor of NO-sensitive guanylyl cyclase, and Ani9, a specific ANO1 channels blocker, had no effect on the luteolin-induced suppression. These results suggest that luteolin inhibited colonic smooth muscle motility by inhibiting L-type calcium channels in mice but not through potassium channels, the enteric nervous system (ENS), NO signaling pathways or ANO1 channels of interstitial cells of Cajal (ICCs).
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Músculo Liso , Animais , Cálcio , Canais de Cálcio Tipo L , Colo , Luteolina , Camundongos , Miócitos de Músculo LisoRESUMO
It is generally considered that enteric neuropathy is one of the causative factors in diabetic gastroparesis. Our previous study demonstrated that there is a loss of NOS neurons in diabetic mice. However, the underlying mechanism remains unclear. The present study was designed to clarify the relationship between neuronal P2X7R and NOS neuron damage. The effect of P2X7R on diabetes-induced gastric NOS neurons damage and its mechanism were investigated by using quantitative RT-PCRï¼immunofluorescence, western blot, isometric force recording, intracellular calcium ([Ca2+]i) measurement and whole-cell patch clamp techniques. The immunohistochemistry and western blot results showed that nNOS expression was significantly down-regulated in diabetic mice, meanwhile, electric field stimulation-induced NOS sensitive relaxation was significantly suppressed. Myenteric neurons expressed P2X7R and pannexin1, and the mRNA and protein level of P2X7R and pannexin1 were up-regulated in diabetic mice. BzATP, a P2X7R activator, evoked [Ca2+]i increase in Hek293 cells with heterologous expression of P2X7R (Hek293-P2X7R cells) and the same dose of ATP-induced [Ca2+]i was more obvious in Hek293-P2X7R cells than in Hek293 cells. Application of BzATP activated an inward current of Hek293-P2X7R in a dose dependent manner. Hek293-P2X7R but not untransfected Hek293 cells could take up of YO-PRO-1. In addition, the uptake of YO-PRO-1 by Hek293-P2X7R was blocked by oxATP, a P2X7 antagonist and CBX, a pannexin1 inhibitor. The results suggest that the P2X7R of enteric neurons may be involved in diabetes-induced NOS neuron damage via combining with pannexin-1 to form transmembrane pores which induce macromolecular substances and calcium into the cells.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Mucosa Gástrica/metabolismo , Neurônios/metabolismo , Óxido Nítrico/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Glicemia/metabolismo , Peso Corporal/efeitos dos fármacos , Cálcio/metabolismo , Fundo Gástrico/efeitos dos fármacos , Fundo Gástrico/metabolismo , Mucosa Gástrica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Neurônios/efeitos dos fármacos , Neurônios/patologia , Óxido Nítrico Sintase Tipo I/metabolismoRESUMO
AIM: To investigate the distribution and function of interstitial cells of Cajal (ICCs) and platelet-derived growth factor receptor-α positive (PDGFRα+) cells in the proximal and distal colon. METHODS: The comparison of colonic transit in the proximal and distal ends was performed by colonic migrating motor complexes (CMMCs). The tension of the colonic smooth muscle was examined by smooth muscle spontaneous contractile experiments with both ends of the smooth muscle strip tied with a silk thread. Intracellular recordings were used to assess electrical field stimulation (EFS)-induced inhibitory junction potentials (IJP) on the colonic smooth muscle. Western blot analysis was used to examine the expression levels of ICCs and PDGFRα in the colonic smooth muscle. RESULTS: Treatment with NG-nitro-L-arginine methyl ester hydrochloride (L-NAME) significantly increased the CMMC frequency and spontaneous contractions, especially in the proximal colon, while treatment with MRS2500 increased only distal CMMC activity and smooth muscle contractions. Both CMMCs and spontaneous contractions were markedly inhibited by NPPB, especially in the proximal colon. Accordingly, CyPPA sharply inhibited the distal contraction of both CMMCs and spontaneous contractions. Additionally, the amplitude of stimulation-induced nitric oxide (NO)/ICC-dependent slow IJPs (sIJPs) by intracellular recordings from the smooth muscles in the proximal colon was larger than that in the distal colon, while the amplitude of electric field stimulation-induced purinergic/PDGFRα-dependent fast IJPs (fIJPs) in the distal colon was larger than that in the proximal colon. Consistently, protein expression levels of c-Kit and anoctamin-1 (ANO1) in the proximal colon were much higher, while protein expression levels of PDGFRα and small conductance calcium-activated potassium channel 3 (SK3) in the distal colon were much higher. CONCLUSION: The ICCs are mainly distributed in the proximal colon and there are more PDGFRα+ cells are in the distal colon, which generates a pressure gradient between the two ends of the colon to propel the feces to the anus.
Assuntos
Colo/citologia , Motilidade Gastrointestinal/fisiologia , Células Gigantes/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Colo/fisiologia , Células Intersticiais de Cajal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismoRESUMO
NEW FINDINGS: What is the central question of this study? The present study investigated the relationship between H2 S and NO in regulation of gastric fundus tension. What is the main finding and its importance? Endogenous or exogenous H2 S and NO have opposite effects on fundus tension, and H2 S-induced gastric fundus tension enhancements are mediated by inhibition of NO generation through the phosphoinositide 3-kinase/Akt pathway. These results are very important in exploring the mechanism of physiological accommodation and accommodation disorder. Hydrogen sulphide (H2 S) is considered a new gasotransmitter, along with NO and CO. It was recently confirmed that H2 S and NO play important roles in the regulation of gastrointestinal smooth muscle tension. The present study was designed to elucidate the interactions between H2 S and NO with respect to the regulation of gastric fundus smooth muscle tension using Western blotting, physiological and electrochemical techniques. Real-time H2 S and NO generation was detected in gastric smooth muscle tissue. NaHS, an H2 S donor, enhanced fundus smooth muscle tension, whereas SNP, an NO donor, decreased fundus smooth muscle tension in a dose-dependent manner. NaHS-induced increases in fundus smooth muscle tension were suppressed by l-NAME, an NO synthase inhibitor. Aminooxyacetic acid (AOAA), a cystathionine ß-synthase inhibitor, exerted inhibitory effects on fundus smooth muscle tension; these effects were also suppressed by l-NAME. Real-time NO generation was significantly potentiated by AOAA. Endothelial nitric oxide synthase (eNOS) phosphorylation at serine 1177 and Akt phosphorylation at serine 308 and threonine 473 were significantly inhibited by NaHS. LY294002, a phosphoinositide 3-kinase inhibitor, blocked these NaHS-mediated effects. However, eNOS phosphorylation at serine 1177 and Akt phosphorylation at serine 308 and threonine 473 were significantly potentiated by AOAA. Cystathionine ß-synthase siRNA interference significantly increased eNOS phosphorylation at serine 1177 and Akt phosphorylation at serine 308 and threonine 473. Cystathionine ß-synthase siRNA interference also increased total eNOS protein expression levels but did not significantly change total Akt kinase protein expression levels. These results suggest that H2 S-induced enhancement of gastric fundus tension is mediated by inhibition of NO generation through the phosphoinositide 3-kinase/Akt pathway.
Assuntos
Fundo Gástrico/efeitos dos fármacos , Sulfeto de Hidrogênio/farmacologia , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Transdução de Sinais , Animais , Masculino , Camundongos , Tono Muscular/efeitos dos fármacos , Músculo Liso/fisiologia , Óxido Nítrico Sintase Tipo III/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Gastrointestinal smooth muscle layer contains two kinds of interstitial cells with special differentiation, i.e., interstitial cells of Cajal (ICC) and platelet-derived growth factor receptor α-positive (PDGFRα+) cells. The ICC and PDGFRα+ cells contact with smooth muscle cells (SMCs) by gap junctions and regulate contractive function of the SMCs. Therefore, these three kinds of cells constitute a functional syncytium, i.e., the SMC, ICC and PDGFRα+ cells syncytium (SIP syncytium). Various neurotransmitters, humoral factors, endogenous bioactive molecules, as well as drugs regulate gastrointestinal motility through the SIP syncytium. In this review, we introduce the concept of SIP syncytium and summarize functions of the syncytium, as well as its physiological and pathological significances.
Assuntos
Motilidade Gastrointestinal , Músculo Liso , Células Gigantes , Humanos , Células Intersticiais de Cajal , Miócitos de Músculo Liso , Receptor alfa de Fator de Crescimento Derivado de PlaquetasRESUMO
Myometrial relaxation of mouse via expression of two-pore domain acid sensitive (TASK) channels was studied. In our previous report, we suggested that two-pore domain acid-sensing K(+) channels (TASK-2) might be one of the candidates for the regulation of uterine circular smooth muscles in mice. In this study, we tried to show the mechanisms of relaxation via TASK-2 channels in marine myometrium. Isometric contraction measurements and patch clamp technique were used to verify TASK conductance in murine myometrium. Western blot and immunehistochemical study under confocal microscopy were used to investigate molecular identity of TASK channel. In this study, we showed that TEA and 4-AP insensitive non-inactivating outward K(+) current (NIOK) may be responsible for the quiescence of murine pregnant longitudinal myometrium. The characteristics of NIOK coincided with two-pore domain acid-sensing K(+) channels (TASK-2). NIOK in the presence of K(+) channel blockers was inhibited further by TASK inhibitors such as quinidine, bupivacaine, lidocaine, and extracellular acidosis. Furthermore, oxytocin and estrogen inhibited NIOK in pregnant myometrium. When compared to non-pregnant myometrium, pregnant myometrium showed stronger inhibition of NIOK by quinidine and increased immunohistochemical expression of TASK-2. Finally, TASK-2 inhibitors induced strong myometrial contraction even in the presence of L-methionine, a known inhibitor of stretch-activated channels in the longitudinal myometrium of mouse. Activation of TASK-2 channels seems to play an essential role for relaxing uterus during pregnancy and it might be one of the alternatives for preventing preterm delivery.
RESUMO
ATP-sensitive potassium (KATP) channels are well characterized in cardiac, pancreatic and many other muscle cells. In the present study, functional expression of the KATP channel was examined in non-pregnant murine longitudinal myometrium. Isometric contraction measurements and Western blot were used. KATP channel openers (KCOs), such as pinacidil, cromakalim, diazoxide and nicorandil, inhibited spontaneous myometrial contractions in a reversible and glibenclamide-sensitive manner. KCOs inhibited oxytocin (OXT)- and prostaglandin F2α (PGF2α)-induced phasic contractions in a glibenclamide-sensitive manner. SUR2B and Kir6.2 were detected by Western blot, whereas SUR1, SUR2A and Kir6.1 were not. These results show that pinacidl, cromakalim, diazoxide and nicorandil-sensitive KATP channels exist in murine myometrium, which are composed of SUR2B and Kir6.2. Based on the modulatory effects of the KATP channel on spontaneous contraction, OXT- and PGF2α-induced contractions, KATP channels seem to play an essential role in murine myometrial motility via activation of SUR2B and Kir6.2.
Assuntos
Canais KATP/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Receptores de Sulfonilureias/metabolismo , Contração Uterina , Trifosfato de Adenosina/metabolismo , Animais , Dinoprosta/antagonistas & inibidores , Feminino , Técnicas In Vitro , Contração Isométrica , Camundongos Endogâmicos ICR , Nicorandil/farmacologia , Potássio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/efeitos dos fármacos , Contração Uterina/efeitos dos fármacosRESUMO
AIM: To investigate the effect of hydrogen sulfide (H2S) on smooth muscle motility in the gastric fundus. METHODS: The expression of cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE) in cultured smooth muscle cells from the gastric fundus was examined by the immunocytochemistry technique. The tension of the gastric fundus smooth muscle was recorded by an isometric force transducer under the condition of isometric contraction with each end of the smooth muscle strip tied with a silk thread. Intracellular recording was used to identify whether hydrogen sulfide affects the resting membrane potential of the gastric fundus in vitro. Cells were freshly separated from the gastric fundus of mice using a variety of enzyme digestion methods and whole-cell patch-clamp technique was used to find the effects of hydrogen sulfide on voltage-dependent potassium channel and calcium channel. Calcium imaging with fura-3AM loading was used to investigate the mechanism by which hydrogen sulfide regulates gastric fundus motility in cultured smooth muscle cells. RESULTS: We found that both CBS and CSE were expressed in the cultured smooth muscle cells from the gastric fundus and that H2S increased the smooth muscle tension of the gastric fundus in mice at low concentrations. In addition, nicardipine and aminooxyacetic acid (AOAA), a CBS inhibitor, reduced the tension, whereas Nω-nitro-L-arginine methyl ester, a nonspecific nitric oxide synthase, increased the tension. The AOAA-induced relaxation was significantly recovered by H2S, and the NaHS-induced increase in tonic contraction was blocked by 5 mmol/L 4-aminopyridine and 1 µmol/L nicardipine. NaHS significantly depolarized the membrane potential and inhibited the voltage-dependent potassium currents. Moreover, NaHS increased L-type Ca(2+) currents and caused an elevation in intracellular calcium ([Ca(2+)]i). CONCLUSION: These findings suggest that H2S may be an excitatory modulator in the gastric fundus in mice. The excitatory effect is mediated by voltage-dependent potassium and L-type calcium channels.
Assuntos
Agonistas dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Fundo Gástrico/efeitos dos fármacos , Motilidade Gastrointestinal/efeitos dos fármacos , Sulfeto de Hidrogênio/farmacologia , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Animais , Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Cistationina gama-Liase/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Fundo Gástrico/metabolismo , Liases/antagonistas & inibidores , Liases/metabolismo , Masculino , Potenciais da Membrana , Camundongos Endogâmicos ICR , Músculo Liso/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Fatores de TempoRESUMO
Our previous studies have shown that CNP-NPR-B/pGC-cGMP is upregulated in the diabetic rats. The present study was designed to determine whether the upregulation of CNP-NPR-B/pGC-cGMP signal pathway affects cGMP-PDE3-cAMP signal pathway in diabetic gastric smooth muscle. The gastric smooth muscle motility was observed by using isometric measurement. PDEs expressions in diabetic gastric smooth muscle tissue were observed by using immunohistochemistry, Western blotting, and RT-PCR methods. The results demonstrated that the inhibitory effect of CNP on the spontaneous contraction of gastric antral circular smooth muscle was potentiated in STZ-induced diabetic rat. CNP-induced increase of cGMP and cAMP was much higher in diabetic gastric smooth muscle tissue than in controls. The expression of PDE3 is downregulated while the levels of gene expression of PDE1, PDE2, PDE4, and PDE5 were not altered in the diabetic gastric smooth muscle tissue. The results suggest that the sensitivity of gastric smooth muscle to CNP is potentiated via activation of CNP-pGC-cGMP-PDE3-cAMP signal pathway in STZ-induced diabetic rats, which may be associated with diabetes-induced gastric motility disorder.
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This study was designed to examine the effects of histamine on gastric motility and its specific receptor in the circular smooth muscle of the human gastric corpus. Histamine mainly produced tonic relaxation in a concentration-dependent and reversible manner, although histamine enhanced contractility in a minor portion of tissues tested. Histamine-induced tonic relaxation was nerve-insensitive because pretreatment with nerve blockers cocktail (NBC) did not inhibit relaxation. Additionally, K(+) channel blockers, such as tetraethylammonium (TEA), apamin (APA), and glibenclamide (Glib), had no effect. However, N(G)-nitro-L-arginine methyl ester (L-NAME) and 1H-(1,2,4)oxadiazolo (4,3-A) quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase (sGC), did inhibit histamine-induced tonic relaxation. In particular, histamine-induced tonic relaxation was converted to tonic contraction by pretreatment with L-NAME. Ranitidine, the H2 receptor blocker, inhibited histamine-induced tonic relaxation. These findings suggest that histamine produced relaxation in circular smooth muscle of human gastric smooth muscle through H2 receptor and NO/sGC pathways.
RESUMO
AIM: To investigate the relationship between neuronal nitric oxide synthase (nNOS) expression and the natriuretic peptide signaling pathway in the gastric fundus of streptozotocin (STZ)-induced diabetic mice. METHODS: Diabetic mice were induced by injection of STZ solution. Immunofluorescence labeling of HuC/D, nNOS and natriuretic peptide receptor-A, B, C (NPRs) in the gastric fundus (GF) was used to observe nNOS expression and whether NPRs exist on enteric neurons. The expression levels of nNOS and NPRs in the diabetic GF were examined by western blotting. An isometric force transducer recorded the electric field stimulation (EFS)-induced relaxation and contraction in the diabetic GF. An intracellular recording method assessed EFS-induced inhibitory junction potentials (IJP) on the GF. GF smooth muscles acquired from normal mice were incubated with different concentrations of the NPRs agonist C-type natriuretic peptide (CNP) for 24 h, after which their nNOS expressions were detected by western blotting. RESULTS: Eight weeks after injection, 43 diabetic mice were obtained from mouse models injected with STZ. Immunofluorescence indicated that the number of NOS neurons was significantly decreased and that nNOS expression was significantly downregulated in the diabetic GF. The results of physiological and electrophysiological assays showed that the EFS-induced relaxation that mainly caused by NO was significantly reduced, while the contraction was enhanced in the diabetic GF. EFS-induced IJP showed that L-NAME sensitive IJP in the diabetic GF was significantly reduced compared with control mice. However, both NPR-A and NPR-B were detected on enteric neurons, and their expression levels were upregulated in the diabetic GF. The nNOS expression level was downregulated dose-dependently in GF smooth muscle tissues exposed to CNP. CONCLUSION: These findings suggested that upregulation of the NPs signaling pathway may be involved in GF neuropathy caused by diabetes by decreasing nNOS expression.
Assuntos
Diabetes Mellitus Experimental/enzimologia , Sistema Nervoso Entérico/enzimologia , Fundo Gástrico/inervação , Músculo Liso/inervação , Peptídeos Natriuréticos/metabolismo , Neurônios Nitrérgicos/enzimologia , Óxido Nítrico Sintase Tipo I/metabolismo , Animais , Glicemia/metabolismo , Peso Corporal , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/fisiopatologia , Regulação para Baixo , Estimulação Elétrica , Sistema Nervoso Entérico/fisiopatologia , Masculino , Camundongos Endogâmicos ICR , Contração Muscular , Óxido Nítrico/metabolismo , Receptores do Fator Natriurético Atrial/metabolismo , Transdução de Sinais , Estreptozocina , Técnicas de Cultura de Tecidos , Regulação para CimaRESUMO
Partial obstruction of the small intestine causes obvious hypertrophy of smooth muscle cells and motility disorder in the bowel proximate to the obstruction. To identify electric remodeling of hypertrophic smooth muscles in partially obstructed murine small intestine, the patch-clamp and intracellular microelectrode recording methods were used to identify the possible electric remodeling and Western blot, immunofluorescence and immunoprecipitation were utilized to examine the channel protein expression and phosphorylation level changes in this research. After 14 days of obstruction, partial obstruction caused obvious smooth muscle hypertrophy in the proximally located intestine. The slow waves of intestinal smooth muscles in the dilated region were significantly suppressed, their amplitude and frequency were reduced, whilst the resting membrane potentials were depolarized compared with normal and sham animals. The current density of voltage dependent potassium channel (KV) was significantly decreased in the hypertrophic smooth muscle cells and the voltage sensitivity of KV activation was altered. The sensitivity of KV currents (IKV) to TEA, a nonselective potassium channel blocker, increased significantly, but the sensitivity of IKv to 4-AP, a KV blocker, stays the same. The protein levels of KV4.3 and KV2.2 were up-regulated in the hypertrophic smooth muscle cell membrane. The serine and threonine phosphorylation levels of KV4.3 and KV2.2 were significantly increased in the hypertrophic smooth muscle cells. Thus this study represents the first identification of KV channel remodeling in murine small intestinal smooth muscle hypertrophy induced by partial obstruction. The enhanced phosphorylations of KV4.3 and KV2.2 may be involved in this process.
Assuntos
Obstrução Intestinal/fisiopatologia , Ativação do Canal Iônico , Músculo Liso/fisiopatologia , Canais de Potássio Shab/metabolismo , Canais de Potássio Shal/metabolismo , 4-Aminopiridina/farmacologia , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Capacitância Elétrica , Imunofluorescência , Secções Congeladas , Hipertrofia , Ativação do Canal Iônico/efeitos dos fármacos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos Endogâmicos ICR , Músculo Liso/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Subunidades Proteicas/metabolismo , Tetraetilamônio/farmacologiaRESUMO
NO and H2S are gaseous signaling molecules that modulate smooth muscle motility. We aimed to identify expressions of enzymes that catalyze H2S and NO generation in mouse gastric smooth muscle, and determine relationships between endogenous H2S and NO in regulation of smooth muscle motility. Western blotting and immunocytochemistry methods were used to track expressions of neuronal nitric oxide synthase (nNOS), endothelial nitric oxide synthase (eNOS), cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE) in gastric smooth muscles. Smooth muscle motility was recorded by isometric force transducers. cGMP production was measured by a specific radioimmunoassay. We found that CBS, CSE, eNOS, and nNOS were all expressed in mice gastric antral smooth muscle tissues, and in cultured gastric antral smooth muscle cells. AOAA significantly inhibited smooth muscle contractions in the gastric antrum, which was significantly recovered by NaHS, while PAG had no significant effect. l-NAME enhanced contractions. NaHS at low concentrations increased basal tension but decreased it at high concentrations. SNP significantly inhibited the contractions, which could be recovered by NaHS both in the absence and presence of CuSO4. ODQ did not block NaHS-induced excitatory effect, while IBMX partially blocked this effect. cGMP production in smooth muscle was significantly increased by SNP but was not affected by NaHS. All these results suggest that endogenous H2S and NO appear to play opposite roles in regulating gastric motility and their effects may be via separate signal transduction pathways. Intracellular H2S/NO levels may be maintained in a state of balance to warrant normal smooth muscle motility.
Assuntos
Motilidade Gastrointestinal/efeitos dos fármacos , Sulfeto de Hidrogênio/farmacologia , Óxido Nítrico/farmacologia , Alcinos/farmacologia , Ácido Amino-Oxiacético/farmacologia , Animais , Cistationina beta-Sintase/antagonistas & inibidores , Cistationina beta-Sintase/fisiologia , Cistationina gama-Liase/antagonistas & inibidores , Cistationina gama-Liase/fisiologia , Glicina/análogos & derivados , Glicina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , NG-Nitroarginina Metil Éster/farmacologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/fisiologia , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/fisiologia , Nitroprussiato/farmacologia , Estômago/efeitos dos fármacos , Estômago/fisiologia , Sulfetos/farmacologiaRESUMO
Plasma pH can be altered during pregnancy and at labor. Membrane excitability of smooth muscle including uterine muscle is suppressed by the activation of K(+) channels. Because contractility of uterine muscle is regulated by extracellular pH and humoral factors, K(+) conductance could be connected to factors regulating uterine contractility during pregnancy. Here, we showed that TASK-2 inhibitors such as quinidine, lidocaine, and extracellular acidosis produced contraction in uterine circular muscle of mouse. Furthermore, contractility was significantly increased in pregnant uterine circular muscle than that of non-pregnant muscle. These patterns were not changed even in the presence of tetraetylammonium (TEA) and 4-aminopyridine (4-AP). Finally, TASK-2 inhibitors induced strong myometrial contraction even in the presence of L-methionine, a known inhibitor of stretchactivated channels in myometrium. When compared to non-pregnant myometrium, pregnant myometrium showed increased immunohistochemical expression of TASK-2. Therefore, TASK-2, seems to play a key role during regulation of myometrial contractility in the pregnancy and provides new insight into preventing preterm delivery.