RESUMEN
PURPOSE: To determine the role of Ca2+ in proliferation,migration and osteogenic differentiation of human dental follicle cells(hDFCs). METHODS: hDFCs were isolated and cultured. The source of hDFCs was detected by immunofluorescence staining. Osteogenesis and adipogenic differentiation of hDFCs was detected by alizarin red staining and oil red O staining, to identify its multi-directional differentiation ability. A series of Ca2+ solutions with different concentrations was prepared, CCK8 assay was used to detect the proliferative abilities at 1, 3, 5, and 7 dï¼migratory ability of 24 h was detected by Transwell assay. Calcium nodules were detected by semiquantitative analysis of alizarin red staining. mRNA expression of osteogenic differentiation related genes was examined by real-time quantitative polymerase chain reaction (RT-qPCR).Statistical analysis was performed using SPSS 17.0 software package. RESULTS: Compared with the control group, 3,4 and 5 mmol/L Ca2+ significantly promoted proliferation of hDFCs at 3, 5 and 7 d (P<0.05). 3, 4, 5 and 6 mmol/L Ca2+ significantly promoted the migration of hDFCs at 24 h(P<0.01). High concentration of Ca2+ had no significant effect on its proliferation and migration. The results of alizarin red staining showed that when Ca2+ concentration reached 4 mmol/L, formation of mineralized nodules were increased(P<0.01), and Ca2+ concentration-dependent. RT-qPCR results showed that Ca2+ up-regulated the expression of RUNX2 and OCN in osteogenic differentiation genes (P<0.01). CONCLUSIONS: Low Ca2+ concentration is beneficial to proliferation and migration, and high Ca2+ concentration is beneficial to osteogenic differentiation of human dental follicle cells.
Asunto(s)
Saco Dental , Osteogénesis , Calcio , Diferenciación Celular , Proliferación Celular , Células Cultivadas , HumanosRESUMEN
BACKGROUND AND PURPOSE: The exact mechanism of protease-activated receptors (PARs) on pacemaker activity of interstitial cells of Cajal (ICCs) has not been reported. We investigated the effects on pacemaker activity by the activation of PARs and its signal mechanisms in colonic ICCs. METHODS: The whole-cell patch-clamp technique, RT-PCR and Ca2+ imaging were used in cultured ICCs from mouse colon. RESULTS: PAR-1 and PAR-2 were expressed in Ano-1 positive ICCs. TFLLR-NH2 (a PAR-1 agonist) and trypsin (a PAR-2 agonist) depolarized the membrane and increased the pacemaker potential frequency. U-73122 (a phospholipase C (PLC) inhibitor) and thapsigargin (a Ca2+ ATPase inhibitor) suppressed the TFLLR-NH2- and trypsin-induced effects on pacemaker potential. TFLLR-NH2 and trypsin also increased intracellular Ca2+ ([Ca2+]i) intensity with increasing of Ca2+ oscillations. Genistein (a tyrosine kinase inhibitor), SP600125 (a JNK inhibitor), CsCl, ZD7288, clonidine (hyperpolarization-activated cyclic nucleotide (HCN) channel blockers), SQ-22536 and dideoxyadenosine (adenylate cyclase inhibitors) suppressed the increased pacemaker potential frequency without effects on depolarization of the membrane induced by TFLLR-NH2 and trypsin. CONCLUSION: These results suggest that activation of PAR-1 and PAR-2 modulates the pacemaker activity of colonic ICCs through the PLC-dependent [Ca2+]i release pathway. The increased pacemaker potential frequency by PAR-1 and PAR-2 was also dependent on tyrosine kinase, JNK, and HCN activation.
Asunto(s)
Colon/fisiología , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/fisiología , Células Intersticiales de Cajal/fisiología , Receptor PAR-1/metabolismo , Receptor PAR-2/metabolismo , Animales , Colon/citología , Colon/efectos de los fármacos , Femenino , Células Intersticiales de Cajal/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Oligopéptidos/farmacología , Técnicas de Placa-Clamp , Receptor PAR-1/agonistas , Receptor PAR-2/agonistas , Receptores Proteinasa-Activados/metabolismoRESUMEN
BACKGROUND: Hyperpolarization-activated cyclic nucleotide (HCN) channels are pacemaker channels that regulate heart rate and neuronal rhythm in spontaneously active cardiac and neuronal cells. Interstitial cells of Cajal (ICCs) are also spontaneously active pacemaker cells in the gastrointestinal tract. Here, we investigated the existence of HCN channel and its role on pacemaker activity in colonic ICCs. METHODS: We performed whole-cell patch clamp, RT-PCR, and Ca(2+)-imaging in cultured ICCs from mouse mid colon. RESULTS: SQ-22536 and dideoxyadenosine (adenylate cyclase inhibitors) decreased the frequency of pacemaker potentials, whereas both rolipram (cAMP-specific phosphodiesterase inhibitor) and cell-permeable 8-bromo-cAMP increased the frequency of pacemaker potentials. CsCl, ZD7288, zatebradine, clonidine (HCN channel blockers), and genistein (a tyrosine kinase inhibitor) suppressed the pacemaker activity. RT-PCR revealed expression of HCN1 and HCN3 channels in c-kit and Ano1 positive colonic ICCs. In recordings of spontaneous intracellular Ca(2+) [Ca(2+)]i oscillations, rolipram and 8-bromo-cAMP increased [Ca(2+)]i oscillations, whereas SQ-22536, CsCl, ZD7288, and genistein decreased [Ca(2+)]i oscillations. CONCLUSIONS: HCN channels in colonic ICCs are tonically activated by basal cAMP production and participate in regulation of pacemaking activity.
Asunto(s)
Calcio/metabolismo , Colon/metabolismo , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Células Intersticiales de Cajal/metabolismo , Animales , Células Cultivadas , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/efectos de los fármacos , Células Intersticiales de Cajal/efectos de los fármacos , Ratones , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
AIM: To investigate lipopolysaccharide (LPS) related signal transduction in interstitial cells of Cajal (ICCs) from mouse small intestine. METHODS: For this study, primary culture of ICCs was prepared from the small intestine of the mouse. LPS was treated to the cells prior to measurement of the membrane currents by using whole-cell patch clamp technique. Immunocytochemistry was used to examine the expression of the proteins in ICCs. RESULTS: LPS suppressed the pacemaker currents of ICCs and this could be blocked by AH6809, a prostaglandin E2-EP2 receptor antagonist or NG-Nitro-L-arginine Methyl Ester, an inhibitor of nitric oxide (NO) synthase. Toll-like receptor 4, inducible NO synthase or cyclooxygenase-2 immunoreactivity by specific antibodies was detected on ICCs. Catalase (antioxidant agent) had no action on LPS-induced action in ICCs. LPS actions were blocked by nuclear factor κB (NF-κB) inhibitor, actinomycin D (a gene transcription inhibitor), PD 98059 (a p42/44 mitogen-activated protein kinases inhibitor) or SB 203580 [a p38 mitogen-activated protein kinases (MAPK) inhibitor]. SB 203580 also blocked the prostaglandin E2-induced action on pacemaker currents in ICCs but not NO. CONCLUSION: LPS inhibit the pacemaker currents in ICCs via prostaglandin E2- and NO-dependent mechanism through toll-like receptor 4 and suggest that MAPK and NF-κB are implicated in these actions.
Asunto(s)
Relojes Biológicos/efectos de los fármacos , Células Intersticiales de Cajal/efectos de los fármacos , Intestino Delgado/efectos de los fármacos , Lipopolisacáridos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , FN-kappa B/metabolismo , Periodicidad , Animales , Antioxidantes/farmacología , Células Cultivadas , Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa 2/farmacología , Dinoprostona/metabolismo , Femenino , Células Intersticiales de Cajal/enzimología , Intestino Delgado/citología , Intestino Delgado/enzimología , Masculino , Potenciales de la Membrana , Ratones , Ratones Endogámicos BALB C , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/metabolismo , Cultivo Primario de Células , Inhibidores de Proteínas Quinasas/farmacología , Receptor Toll-Like 4/efectos de los fármacos , Receptor Toll-Like 4/metabolismoRESUMEN
Interstitial cells of Cajal (ICC) are the pacemaker cells that generate the rhythmic oscillation responsible for the production of slow waves in gastrointestinal smooth muscle. Spingolipids are known to present in digestive system and are responsible for multiple important physiological and pathological processes. In this study, we are interested in the action of sphingosine 1-phosphate (S1P) on ICC. S1P depolarized the membrane and increased tonic inward pacemaker currents. FTY720 phosphate (FTY720P, an S1P(1,3,4,5) agonist) and SEW 2871 (an S1P(1) agonist) had no effects on pacemaker activity. Suramin (an S1P(3) antagonist) did not block the S1P-induced action on pacemaker currents. However, JTE-013 (an S1P(2) antagonist) blocked the S1P-induced action. RT-PCR revealed the presence of the S1P(2) in ICC. Calphostin C (a protein kinase C inhibitor), NS-398 (a cyclooxygenase-2 inhibitor), PD 98059 (a p42/44 inhibitor), or SB 203580 (a p38 inhibitor) had no effects on S1P-induced action. However, c-jun NH(2)-terminal kinase (JNK) inhibitor II suppressed S1P-induced action. External Ca(2+)-free solution or thapsigargin (a Ca(2+)-ATPase inhibitor of endoplasmic reticulum) suppressed action of S1P on ICC. In recording of intracellular Ca(2+) ([Ca(2+)](i)) concentration using fluo-4/AM S1P increased intensity of spontaneous [Ca(2+)](i) oscillations in ICC. These results suggest that S1P can modulate pacemaker activity of ICC through S1P(2) via regulation of external and internal Ca(2+) and mitogenactivated protein kinase activation.
Asunto(s)
Células Intersticiales de Cajal/efectos de los fármacos , Intestino Delgado/efectos de los fármacos , Canales Iónicos/efectos de los fármacos , Lisofosfolípidos/farmacología , Receptores de Lisoesfingolípidos/metabolismo , Transducción de Señal/efectos de los fármacos , Esfingosina/análogos & derivados , Animales , Antinematodos/farmacología , Calcio/metabolismo , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Femenino , Células Intersticiales de Cajal/citología , Células Intersticiales de Cajal/metabolismo , Intestino Delgado/citología , Intestino Delgado/metabolismo , Canales Iónicos/metabolismo , Masculino , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Naftalenos/metabolismo , Organofosfatos/farmacología , Técnicas de Placa-Clamp , Pirazoles/farmacología , Piridinas/farmacología , Receptores de Lisoesfingolípidos/genética , Esfingosina/farmacología , Receptores de Esfingosina-1-Fosfato , Suramina/farmacologíaRESUMEN
BACKGROUND AND PURPOSE: Lipopolysaccharide (LPS) induces intestinal dysmotility by alteration of smooth muscle and enteric neuronal activities. However, there is no report on the modulatory effects of LPS on the interstitial cells of Cajal (ICCs). We investigated the effect of LPS and its signal transduction in ICCs. METHODS: We performed whole-cell patch clamp and RT-PCR in cultured ICCs from mouse small intestine. RESULTS: LPS suppressed the generation of pacemaker currents of ICCs. The mRNA transcripts for Toll-like receptor 4 (TLR4) were expressed in ICCs. However, the inhibitory action of LPS on pacemaker currents from TLR4(+/+) mice was not present in TLR4(-/-) mice. The inhibitory effects of LPS on ICCs were blocked by glibenclamide (an inhibitor of ATP-sensitive K(+) channels), NS-398 (a COX-2 inhibitor), AH6808 [a prostaglandin E(2) (PGE(2))-EP(2) receptor antagonist], ODQ (an inhibitor of guanylate cyclase) and L-NAME [an inhibitor of nitric oxide synthase (NOS)]. Furthermore, genistein and herbimycin A (tyrosine kinase inhibitors) blocked the LPS-induced inhibitory action on pacemaker activity in ICCs. CONCLUSIONS: LPS can activate ICCs to release NO and PGE(2) through TLR4 activation. The released NO and PGE(2) inhibit pacemaker currents by activating ATP-sensitive K(+) channels. The LPS actions are mediated by tyrosine kinase signaling pathways.
Asunto(s)
Células Intersticiales de Cajal/efectos de los fármacos , Lipopolisacáridos/farmacología , Animales , Dinoprostona/biosíntesis , Células Intersticiales de Cajal/fisiología , Canales KATP/fisiología , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico/biosíntesis , Proteínas Tirosina Quinasas/fisiología , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/fisiologíaRESUMEN
Neurotensin, a tridecapeptide localized in the gut to discrete enteroendocrine cells of the small bowel mucosa, is a hormone that plays an important role in gastrointestinal secretion, growth, and motility. Neurotensin has inhibitory and excitatory effects on peristaltic activity and produces contractile and relaxant responses in intestinal smooth muscle. Our objective in this study is to investigate the effects of neurotensin in small intestinal interstitial cells of Cajal (ICC) and elucidate the mechanism. To determine the electrophysiological effects of neurotensin on ICC, whole-cell patch clamp recordings were performed in cultured ICC from the small intestine. Exposure to neurotensin depolarized the membrane of pacemaker cells and produced tonic inward pacemaker currents. Only neurotensin receptor1 was identified when RT-PCR and immunocytochemistry were performed with mRNA isolated from small intestinal ICC and c-Kit positive cells. Neurotensin-induced tonic inward pacemaker currents were blocked by external Naâº-free solution and in the presence of flufenamic acid, an inhibitor of non-selective cation channels. Furthermore, neurotensin-induced action is blocked either by treatment with U73122, a phospholipase C inhibitor, or thapsigargin, a Ca²âº-ATPase inhibitor in ICC. We found that neurotensin increased spontaneous intracellular Ca²âº oscillations as seen with fluo4/AM recording. These results suggest that neurotensin modulates pacemaker currents via the activation of non-selective cation channels by intracellular Ca²âº-release through neurotensin receptor1.
Asunto(s)
Células Intersticiales de Cajal/metabolismo , Intestino Delgado/metabolismo , Músculo Liso/metabolismo , Neurotensina/metabolismo , Neurotensina/farmacología , Animales , Calcio/metabolismo , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , ATPasas Transportadoras de Calcio/metabolismo , Células Cultivadas , Retículo Endoplásmico/metabolismo , Femenino , Técnicas In Vitro , Células Intersticiales de Cajal/efectos de los fármacos , Intestino Delgado/citología , Intestino Delgado/efectos de los fármacos , Canales Iónicos/metabolismo , Masculino , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos BALB C , Receptores de Neurotensina/metabolismo , Sodio/metabolismo , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/metabolismoRESUMEN
We studied whether nitric oxide (NO) and hydrogen sulfide (H(2)S) have an interaction on the pacemaker activities of interstitial cells of Cajal (ICC) from the mouse small intestine. The actions of NO and H(2)S on pacemaker activities were investigated by using the whole-cell patch-clamp technique and intracellular Ca(2+) analysis at 30â in cultured mouse ICC. Exogenously applied (±)-S-nitroso-N-acetylpenicillamine (SNAP), an NO donor, or sodium hydrogen sulfide (NaHS), a donor of H(2)S, showed no influence on pacemaker activity (potentials and currents) in ICC at low concentrations (10 µM SNAP and 100 µM NaHS), but SNAP or NaHS completely inhibited pacemaker amplitude and pacemaker frequency with increases in the resting currents in the outward direction at high concentrations (SNAP 100 µM and NaHS 1 mM). Co-treatment with 10 µM SNAP plus 100 µM NaHS also inhibited pacemaker amplitude and pacemaker frequency with increases in the resting currents in the outward direction. ODQ, a guanylate cyclase inhibitor, or glibenclamide, an ATP-sensitive K(+) channel inhibitor, blocked the SNAP+NaHS-induced inhibition of pacemaker currents in ICC. Also, we found that SNAP+NaHS inhibited the spontaneous intracellular Ca(2+) ([Ca(2+)](i)) oscillations in cultured ICC. In conclusion, this study describes the enhanced inhibitory effects of NO plus H(2)S on ICC in the mouse small intestine. NO+H(2)S inhibited the pacemaker activity of ICC by modulating intracellular Ca(2+). These results may be evidence of a physiological interaction of NO and H(2)S in ICC for modulating gastrointestinal motility.
RESUMEN
In this study we determined whether or not 5-hydroxytryptamine (5-HT) has an effect on the pacemaker activities of interstitial cells of Cajal (ICC) from the mouse small intestine. The actions of 5-HT on pacemaker activities were investigated using a whole-cell patch-clamp technique, intracellular Ca(2+) ([Ca(2+)](i)) analysis, and RT-PCR in ICC. Exogenously-treated 5-HT showed tonic inward currents on pacemaker currents in ICC under the voltage-clamp mode in a dose-dependent manner. Based on RT-PCR results, we found the existence of 5-HT(2B, 3, 4, and 7) receptors in ICC. However, SDZ 205557 (a 5-HT(4) receptor antagonist), SB 269970 (a 5-HT7 receptor antagonist), 3-tropanylindole - 3 - carboxylate methiodide (3-TCM; a 5-HT(3) antagonist) blocked the 5-HT-induced action on pacemaker activity, but not SB 204741 (a 5-HT(2B) receptor antagonist). Based on [Ca(2+)](i) analysis, we found that 5-HT increased the intensity of [Ca(2+)](i). The treatment of PD 98059 or JNK II inhibitor blocked the 5-HT-induced action on pacemaker activity of ICC, but not SB 203580. In summary, these results suggest that 5-HT can modulate pacemaker activity through 5-HT(3, 4, and 7) receptors via [Ca(2+)](i) mobilization and regulation of mitogen-activated protein kinases.
RESUMEN
BACKGROUND/AIMS: Capsaicin (8-methyl-N-vanillyl-6-ninenamide), a compound found in hot peppers, has been reported to have different physiological actions on different cell types. Not much work has been done about the effect of capsaicin on the function of interstitial cells of Cajal (ICC). In the present study, we examined the action of external application of capsaicin on pacemaker activity in the cultured ICC from the small intestine of mouse. METHODS: We investigated the effect of capsaicin on pacemaker currents in cultured ICC from the small intestine of mouse using a whole cell patch-clamp technique and Ca(2+)-imaging analysis. RESULTS: When capsaicin was applied externally to the pacemaker generating ICC, it completely inhibited the pacemaker potential under current-clamp mode (I = 0) and the pacemaker current under voltage-clamp mode at a -70 mV of holding potentials. The effect of capsaicin on pacemaker activity in ICC was shown dose dependently. The effect of capsaicin was not through the transient receptor potential of the vanilloid type 1 (TRPV1) channel as capsazepine did not block the effect of capsaicin. L-NAME, an inhibitor of nitric oxide synthase, also did not block the capsaicin-induced effects. When the action of capsaicin was examined in the intracellular calcium oscillation, it completely abolished the calcium oscillation. CONCLUSIONS: These results prove that the capsaicin has the inhibitory effects on the ICC which is carried out neither through TRPV channel nor the nitric oxide production. Intracellular Ca(2+) was also an important target for actions of capsaicin on ICC.
RESUMEN
The effects of (-)-epigallocatechin gallate (EGCG) on pacemaker activities of cultured interstitial cells of Cajal (ICC) from murine small intestine were investigated using whole-cell patch-clamp technique at 30 and Ca(2+) image analysis. ICC generated spontaneous pacemaker currents at a holding potential of -70 mV. The treatment of ICC with EGCG resulted in a dose-dependent decrease in the frequency and amplitude of pacemaker currents. SQ-22536, an adenylate cyclase inhibitor, and ODQ, a guanylate cyclase inhibitor, did not inhibit the effects of EGCG. EGCG-induced effects on pacemaker currents were not inhibited by glibenclamide, an ATP-sensitive K(+) channel blocker and TEA, a Ca(2+)-activated K(+) channel blocker. Also, we found that EGCG inhibited the spontaneous [Ca(2+)](i) oscillations in cultured ICC. In conclusion, EGCG inhibited the pacemaker activity of ICC and reduced [Ca(2+)](i) oscillations by cAMP-, cGMP-, ATP-sensitive K+ channel-independent manner.