RESUMO
Liver transplantation is the only life-saving treatment for end-stage liver failure but is limited by the organ shortage and consequences of immunosuppression. Repopulation of decellularised scaffolds with recipient cells provides a theoretical solution, allowing reliable and timely organ sourcing without the need for immunosuppression. Recellularisation of the vasculature of decellularised liver scaffolds was investigated as an essential prerequisite to the survival of other parenchymal components. Liver decellularisation was carried out by portal vein perfusion using a detergent-based solution. Decellularised scaffolds were placed in a sterile perfusion apparatus consisting of a sealed organ chamber, functioning at 37°C in normal atmospheric conditions. The scaffold was perfused via portal vein with culture medium. A total of 107 primary cultured bone marrow stem cells, selected by plastic adherence, were infused into the scaffold, after which repopulated scaffolds were perfused for up to 30 days. The cultured stem cells were assessed for key marker expression using fluorescence-activated cell sorting (FACS), and recellularised scaffolds were analysed by light, electron and immunofluorescence microscopy. Stem cells were engrafted in portal, sinusoidal and hepatic vein compartments, with cell alignment reminiscent of endothelium. Cell surface marker expression altered following engraftment, from haematopoietic to endothelial phenotype, and engrafted cells expressed sinusoidal endothelial endocytic receptors (mannose, Fc and stabilin receptors). These results represent one step towards complete recellularisation of the liver vasculature and progress towards the objective of generating transplantable neo-organs.
Assuntos
Células da Medula Óssea/citologia , Fígado/citologia , Veia Porta/citologia , Células-Tronco/citologia , Alicerces Teciduais/química , Animais , Antígenos Comuns de Leucócito/metabolismo , Fígado/ultraestrutura , Masculino , Perfusão , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Ratos Endogâmicos LewRESUMO
Hepatocyte transplantation (HcTx) is a promising approach for the treatment of metabolic diseases in newborns and children. The most common application route is the portal vein, which is difficult to access in the newborn. Transfemoral access to the splenic artery for HcTx has been evaluated in adults, with trials suggesting hepatocyte translocation from the spleen to the liver with a reduced risk for thromboembolic complications. Using juvenile Göttingen minipigs, we aimed to evaluate feasibility of hepatocyte transplantation by transfemoral splenic artery catheterization, while providing insight on engraftment, translocation, viability, and thromboembolic complications. Four Göttingen Minipigs weighing 5.6 kg to 12.6 kg were infused with human hepatocytes (two infusions per cycle, 1.00E08 cells per kg body weight). Immunosuppression consisted of tacrolimus and prednisolone. The animals were sacrificed directly after cell infusion (n=2), 2 days (n=1), or 14 days after infusion (n=1). The splenic and portal venous blood flow was controlled via color-coded Doppler sonography. Computed tomography was performed on days 6 and 18 after the first infusion. Tissue samples were stained in search of human hepatocytes. Catheter placement was feasible in all cases without procedure-associated complications. Repetitive cell transplantations were possible without serious adverse effects associated with hepatocyte transplantation. Immunohistochemical staining has proven cell relocation to the portal venous system and liver parenchyma. However, cells were neither present in the liver nor the spleen 18 days after HcTx. Immunological analyses showed a response of the adaptive immune system to the human cells. We show that interventional cell application via the femoral artery is feasible in a juvenile large animal model of HcTx. Moreover, cells are able to pass through the spleen to relocate in the liver after splenic artery infusion. Further studies are necessary to compare this approach with umbilical or transhepatic hepatocyte administration.
Assuntos
Hepatócitos/transplante , Fígado/citologia , Artéria Esplênica , Animais , Cateterismo/métodos , Transplante de Células/efeitos adversos , Transplante de Células/métodos , Hepatócitos/citologia , Hepatócitos/enzimologia , Hepatócitos/imunologia , Humanos , Terapia de Imunossupressão , Fígado/enzimologia , Fígado/patologia , Modelos Animais , Veia Porta/citologia , Baço/citologia , Baço/diagnóstico por imagem , Baço/patologia , Artéria Esplênica/citologia , Suínos , Porco Miniatura , Fatores de Tempo , Tomografia Computadorizada por Raios X , Ultrassonografia DopplerRESUMO
AIM: Surgical management of pediatric extrahepatic portal vein obstruction requires meso-Rex bypass using autologous or synthetic grafts. Tissue-engineered vascular grafts (TEVGs) provide an alternative, but no validated animal models using portal TEVGs exist. Herein, we preclinically assess TEVGs as portal vein bypass grafts. MATERIALS & METHODS: TEVGs were implanted as portal vein interposition conduits in SCID-beige mice, monitored by ultrasound and micro-computed tomography, and histologically assessed postmortem at 12 months. RESULTS: TEVGs remained patent for 12 months. Histologic analysis demonstrated formation of neovessels that resembled native portal veins, with similar content of smooth muscle cells, collagen type III and elastin. CONCLUSION: TEVGs are feasible portal vein conduits in a murine model. Further preclinical evaluation of TEVGs may facilitate pediatric clinical translation.
Assuntos
Prótese Vascular , Modelos Animais , Veia Porta/citologia , Veia Porta/transplante , Engenharia Tecidual/métodos , Animais , Feminino , Camundongos , Camundongos SCID , Alicerces Teciduais , Microtomografia por Raio-XRESUMO
The hepatic lobule is divided into three zones along the portal-central vein axis. Hepatocytes within each zone exhibit a distinctive gene expression profile that coordinates their metabolic compartmentalization. The zone-dependent heterogeneity of hepatocytes has been hypothesized to result from the differential degree of exposure to oxygen, nutrition and gut-derived toxins. In addition, the gradient of Wnt signaling that increases towards the central vein seen in rodent models is believed to play a critical role in shaping zonation. Furthermore, hepatic zonation is coupled to the site of the homeostatic renewal of hepatocytes. Despite its critical role, the regulatory mechanisms that determine the distinctive features of zonation and its relevance to humans are not well understood. The present study first conducted a comprehensive zone-dependent transcriptome analysis of normal human liver using laser capture microdissection. Upstream pathway analysis revealed the signatures of host responses to gut-derived toxins in the periportal zone, while both the canonical Wnt pathway and the xenobiotic response pathway govern the perivenular zone. Furthermore, we found that the hypoxic environment of the perivenular zone promotes Wnt11 expression in hepatocytes, which then regulates unique gene expression via activation of the non-canonical Wnt pathway. In summary, our study reports the comprehensive zonation-dependent transcriptome of the normal human liver. Our analysis revealed that the LPS response pathway shapes the characteristics of periportal hepatocytes. By contrast, the perivenular zone is regulated by a combination of three distinct pathways: the xenobiotic response pathway, canonical Wnt signaling, and hypoxia-induced noncanonical Wnt signaling.
Assuntos
Fígado/citologia , Veia Porta/citologia , Transcriptoma/genética , Proteínas Wnt/genética , Hipóxia Celular/genética , Linhagem Celular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Hepatócitos/metabolismo , Humanos , Fígado/crescimento & desenvolvimento , Veia Porta/crescimento & desenvolvimento , Veia Porta/metabolismo , Via de Sinalização Wnt/genética , Xenobióticos/metabolismo , beta Catenina/metabolismoRESUMO
Although islet transplantation has been suggested as an alternative therapy for type 1 diabetes, there are efficiency concerns that are attributed to poor engraftment of transplanted islets. Hypoxic condition and delayed vasculogenesis induce necrosis and apoptosis of the transplanted islets. To overcome these limitations in islet transplantation, heterospheroids (HSs), which consist of rat islet cells (ICs) and human bone marrow-derived mesenchymal stem cells (hMSCs), were transplanted to the kidney and liver. The HSs cultured under the hypoxic condition system exhibited a significant increase in antiapoptotic gene expression in ICs. hMSCs in the HSs secreted angiogenic and antiapoptotic proteins. With the HS system, ICs and hMSCs were successfully located in the same area of the liver after transplantation of HSs through the portal vein, whereas the transplantation of islets and the dissociated hMSCs did not result in localization of transplanted ICs and hMSCs in the same area. HS transplantation resulted in an increase in angiogenesis at the transplantation area and a decrease in the apoptosis of transplanted ICs after transplantation into the kidney subcapsule compared with transplantation of islet cell clusters (ICCs). Insulin production levels of ICs were higher in the HS transplantation group compared with the ICC transplantation group. The HS system may be a more efficient transplantation method than the conventional methods for the treatment of type 1 diabetes.
Assuntos
Apoptose , Transplante das Ilhotas Pancreáticas , Ilhotas Pancreáticas/citologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Neovascularização Fisiológica , Esferoides Celulares/citologia , Animais , Agregação Celular , Contagem de Células , Sobrevivência Celular , Células Cultivadas , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Insulina/metabolismo , Rim/citologia , Fígado/citologia , Masculino , Veia Porta/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-DawleyRESUMO
TMEM16A is a major component of Ca(2+)-activated Cl(-) channel (CaCC) conductance in murine portal vein smooth muscle cells (mPVSMCs). Here, the regulation of CaCC activity by the actin cytoskeleton was examined in mPVSMCs. Actin disruption by cytochalasin D did not affect the current density, but increased the deactivation time constant in mPVSMCs. The elongated deactivation was recovered by jasplakinolide. When murine TMEM16A was transfected into HEK293 cells that have a poorly developed actin cytoskeleton, electrophysiological properties of CaCC currents were not changed by cytochalasin D. In conclusion, the CaCC activity in mPVSMCs is modified by the interaction of TMEM16A with abundant actin cytoskeleton.
Assuntos
Citoesqueleto de Actina/fisiologia , Canais de Cloreto/fisiologia , Fenômenos Eletrofisiológicos/genética , Veia Porta/citologia , Animais , Anoctamina-1 , Células Cultivadas , Canais de Cloreto/genética , Citocalasina D/farmacologia , Depsipeptídeos/farmacologia , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Células HEK293 , Humanos , Camundongos , Músculo Liso Vascular , TransfecçãoRESUMO
The use of avian animal models has contributed to the understanding of many aspects of the ontogeny of the hematopoietic system in vertebrates. However, specific events that occur in the model itself are still unclear. There is a lack of consensus, among previous studies, about which is the intermediate site responsible for expansion and differentiation of hematopoietic cells, and the liver's contribution to the development of this system. Here we aimed to evaluate the presence of hematopoiesis in the yolk sac and liver in chickens, from the stages of intra-aortic clusters in the aorta-genital ridges-mesonephros (AGM) region until hatching, and how it relates to the establishment of the bone marrow. Gallus gallus domesticus L. embryos and their respective yolk sacs at embryonic day 3 (E3) and up to E21 were collected and processed according to standard histological techniques for paraffin embedding. The slides were stained with hematoxylin-eosin, Lennert's Giemsa, and Sirius Red at pH 10.2, and investigated by light microscopy. This study demonstrated that the yolk sac was a unique hematopoietic site between E4 and E12. Hematopoiesis occurred in the yolk sac and bone marrow between E13 and E20. The liver showed granulocytic differentiation in the connective tissue of portal spaces at E15 and onwards. The yolk sac showed expansion of erythrocytic and granulocytic lineages from E6 to E19, and E7 to E20, respectively. The results suggest that the yolk sac is the major intermediate erythropoietic and granulopoietic site where expansion and differentiation occur during chicken development. The hepatic hematopoiesis is restricted to the portal spaces and represented by the granulocytic lineage.
Assuntos
Células da Medula Óssea/citologia , Hematopoese , Fígado/citologia , Fígado/embriologia , Saco Vitelino/citologia , Saco Vitelino/embriologia , Animais , Embrião de Galinha , Eritropoese , Granulócitos/citologia , Fígado/irrigação sanguínea , Veia Porta/citologia , Veia Porta/embriologia , Fatores de TempoRESUMO
Ca(2+)-activated Cl(-) channel (CaCC) often plays substantial roles in the regulation of membrane excitability in smooth muscle cells (SMCs). TMEM16A, a member of the TMEM16 family, has been suggested as the molecular entity responsible for CaCC in several types of SMCs. In this study, the expression of TMEM16A splicing variants and their contribution to CaCC activity were examined in murine portal vein SMCs (mPVSMCs). Four transcripts of TMEM16A splicing variants, which include four alternatively spliced segments ("a" and "b" in N-terminus and "c" and "d" in the first intracellular loop), were identified; the expression ratio of four transcripts of "abc", "acd", "abcd" and "ac" was 64.5, 25.8, 4.8 and 4.8%, respectively. The immunostaining of isolated mPVSMCs with anti-TMEM16A antibody indicates the abundant expression of TMEM16A on the cell membrane. CaCC currents recorded in mPVSMCs were markedly reduced by T16A(inh)-A01, a specific TMEM16A inhibitor. When the two major TMEM16A splicing variants, abc and acd isoforms, were expressed separately in HEK293 cells, the CaCC currents, which possess similar electrophysiological characteristics to those in mPVSMCs were observed. The single-molecule photobleaching analyses using total internal reflection fluorescence (TIRF) microscope indicated that the distribution of stepwise photobleaching events was fit well with a binomial distribution for homodimer. Additionally, the heterodimer formation was suggested by fluorescence resonance energy transfer (FRET) analyses in HEK293 cells co-expressing CFP- or YFP-tagged variants. In conclusion, alternatively spliced variants of TMEM16A abc and acd in mPVSMCs are two major molecular entities of CaCC and may form hetero-/homo-dimers to be functional as CaCC in the regulation of membrane excitability and contractility in mPVSMCs.
Assuntos
Canais de Cloreto/metabolismo , Miócitos de Músculo Liso/metabolismo , Veia Porta/metabolismo , Isoformas de Proteínas/metabolismo , Animais , Anoctamina-1 , Células Cultivadas , Dimerização , Regulação da Expressão Gênica/fisiologia , Masculino , Camundongos , Veia Porta/citologiaRESUMO
The mechanical forces acting on SMC in the vascular wall are known to regulate processes such as vascular remodeling and contractile differentiation. However, investigations to elucidate the underlying mechanisms of mechanotransduction in smooth muscle have been hampered by technical limitations associated with mechanical studies on pressurized small arteries, due primarily to the small amount of available tissue. The murine portal vein is a relatively large vessel showing myogenic tone that in many respects recapitulates the properties of small resistance vessels. Studies on stretched portal veins to elucidate mechanisms of mechanotransduction in the vascular wall have shown that stretch-sensitive regulation of contractile differentiation is mediated via Rho-activation and actin polymerization, while stretch-induced growth is regulated by the MAPK pathway. In this review, we have summarized findings on mechanotransduction in the portal vein with focus on stretch-induced contractile differentiation and the role of calcium, actin polymerization and miRNAs in this response.
Assuntos
Actinas/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/fisiologia , Veia Porta/citologia , Veia Porta/fisiologia , Animais , Fenômenos Biomecânicos , Sinalização do Cálcio , Diferenciação Celular , Humanos , Mecanotransdução Celular , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Modelos Cardiovasculares , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/fisiologia , PolimerizaçãoRESUMO
Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 µm pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.
Assuntos
Técnicas Citológicas/métodos , Hepatócitos/citologia , Fígado/citologia , Animais , Técnicas de Cultura de Células/métodos , Fígado/cirurgia , Perfusão , Veia Porta/citologia , Veia Porta/cirurgia , RatosRESUMO
Portal interstitial cells of Cajal (PICCs), acting as vascular pacemakers, were previously only identified in nonhumans. Moreover, there is no evidence available about the presence of such cells within the liver. The objective of the study is to evaluate whether or not PICCs are identifiable in humans and, if they are, whether or not they are following the scaffold of portal vein (PV) branches within the liver. We obtained extrahepatic PVs and liver samples from six adult human cadavers, negative for liver disease, in accordance with ethical rules. They were stained with hematoxylin-eosin (HE) and Giemsa, and then we performed immunohistochemistry on formalin-fixed paraffin-embedded specimens for CD117/c-kit, a marker of the Cajal's cells. Immune labeling was also performed for S-100 protein, desmin, glial fibrillary acidic protein (GFAP), neurofilaments, α-smooth muscle actin (α-SMA), and CD34. c-kit-Positive PICCs were identified within the extrahepatic PV, in portal spaces, and septa. On adjacent sections, these PICCs were negative for all the other antibodies used. In conclusion, our study confirms the presence of extrahepatic PICCs on humans, which may act as a possible intrinsic pacemaker in the human PV. However, the intrahepatic PICCs, which were evidenced here for the first time, are in need for further experimental studies to evaluate their functional role. A promising further direction of the study is the PICCs role in the idiopathic portal hypertension.
Assuntos
Células Intersticiais de Cajal , Fígado/irrigação sanguínea , Veia Porta/citologia , Actinas/análise , Idoso , Antígenos CD34/análise , Biomarcadores/análise , Cadáver , Desmina/análise , Feminino , Proteína Glial Fibrilar Ácida/análise , Humanos , Imuno-Histoquímica , Células Intersticiais de Cajal/química , Masculino , Pessoa de Meia-Idade , Proteínas de Neurofilamentos/análise , Veia Porta/química , Proteínas Proto-Oncogênicas c-kit/análise , Proteínas S100/análise , Coloração e RotulagemRESUMO
Structural organization of hepatic portal vein (HPV) was examined in adult rats by means of light and electron microscopy. Three characteristic features were found in the wall structure of rat HPV. (1) Tunica media consisted of two kinds of smooth muscle. The inner circular smooth muscle (CSM) was composed with one or two layer of smooth muscle cells, and was found in the entire length of the HPV and its tributaries. The outer longitudinal smooth muscle (LSM) was limited to a specific region of HPV; in particular it was well-developed at distal half of HPV. CSM counteracts luminal hydrostatic pressure to prevent circumferential hyperextension of venous wall, whereas LSM is likely to counteract a tractive force produced by gravity and movement of small intestine. (2) Intima of HPV showed a unique feature, intimal folds, which protruded into the lumen and were aligned almost circumferentially. Intimal folds were found only at the same region where the LSM was well-developed. Thus, LSM is presumably involved in the formation of intimal folds. (3) The endothelial cells between intimal folds were circumferentially aligned along the folds, although those in the other regions of HPV were arrayed along the longitudinal axis of HPV or the direction of blood flow as reported in other kinds of blood vessel. This finding implied that the circumferential blood flow locally occurs on the surface of intima between the intimal folds.
Assuntos
Endotélio Vascular/citologia , Fígado/irrigação sanguínea , Músculo Liso Vascular/citologia , Veia Porta/citologia , Túnica Íntima/citologia , Animais , Endotélio Vascular/fisiologia , Endotélio Vascular/ultraestrutura , Masculino , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Músculo Liso Vascular/fisiologia , Músculo Liso Vascular/ultraestrutura , Veia Porta/fisiologia , Veia Porta/ultraestrutura , Ratos , Ratos Wistar , Fluxo Sanguíneo Regional/fisiologia , Túnica Íntima/fisiologia , Túnica Íntima/ultraestrutura , Túnica Média/citologia , Túnica Média/fisiologia , Túnica Média/ultraestruturaRESUMO
Stretch of the vascular wall stimulates smooth muscle hypertrophy by activating the MAPK and Rho/Rho kinase (ROK) pathways. We investigated the role of calcium in this response. Stretch-stimulated expression of contractile and cytoskeletal proteins in mouse portal vein was inhibited at mRNA and protein levels by blockade of voltage-dependent Ca(2+) entry (VDCE). In contrast, blockade of store-operated Ca(2+) entry (SOCE) did not affect smooth muscle marker expression but decreased global protein synthesis. Activation of VDCE caused membrane translocation of RhoA followed by phosphorylation of its downstream effectors LIMK-2 and cofilin-2. Stretch-activated cofilin-2 phosphorylation depended on VDCE but not on SOCE. VDCE was associated with increased mRNA expression of myocardin, myocyte enhancer factor (MEF) -2A and -2D, and smooth muscle marker genes, all of which depended on ROK activity. SOCE increased ERK1/2 phosphorylation and c-Fos expression but had no effect on phosphorylation of LIMK-2 and cofilin-2 or on myocardin and MEF2 expression. Knockdown of MEF2A or -2D eliminated the VDCE-induced activation of myocardin expression and increased basal c-Jun and c-Fos mRNA levels. These results indicate that MEF2 mediates VDCE-dependent stimulation of myocardin expression via the Rho/ROK pathway. In addition, SOCE activates the expression of immediate-early genes, known to be regulated by MEF2 via Ca(2+)-dependent phosphorylation of histone deacetylases, but this mode of Ca(2+) entry does not affect the Rho/ROK pathway. Compartmentation of Ca(2+) entry pathways appears as one mechanism whereby extracellular and membrane signals influence smooth muscle phenotype regulation, with MEF2 as a focal point.
Assuntos
Cálcio/metabolismo , Diferenciação Celular/fisiologia , Membrana Celular/metabolismo , Músculo Liso Vascular/metabolismo , Veia Porta/metabolismo , Transdução de Sinais/fisiologia , Animais , Membrana Celular/genética , Cofilina 2/genética , Cofilina 2/metabolismo , Feminino , Regulação da Expressão Gênica/fisiologia , Técnicas de Silenciamento de Genes , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Quinases Lim/genética , Quinases Lim/metabolismo , Fatores de Transcrição MEF2 , Camundongos , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Músculo Liso Vascular/citologia , Fatores de Regulação Miogênica/genética , Fatores de Regulação Miogênica/metabolismo , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Fosforilação/fisiologia , Veia Porta/citologia , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Transativadores/biossíntese , Transativadores/genética , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTPRESUMO
Geometric measures of localized calcium release (LCR) events have been used to understand their biophysical basis. We found power law scaling between three such metrics-maximum amplitude (MA), mass above half-maximum amplitude (MHM), and area at half-maximum amplitude (AHM). In an effort to understand this scaling a minimal analytic model was employed to simulate LCR events recorded by confocal line scan. The distribution of logMHM as a function of logAHM, pMHM(pAHM), was dependent on model parameters such as channel open time, current size, line scan offset, and apparent diffusion coefficient. The distribution of log[MHM/AHM] as a function of logMA, p[MHM/AHM](pMA), was invariant, reflecting the gross geometry of the LCR event. The findings of the model were applied to real LCR line scan data from rabbit portal vein myocytes, rat cerebral artery myocytes, and guinea pig fundus knurled cells. pMHM(pAHM) could be used to distinguish two populations of LCR events in portal vein, even at the scale of "calcium noise," and to calculate the relative current of the two. The relative current was 2. pMHM(pAHM) could also be used to study pharmacological effects. The pMHM(pAHM) distribution of knurled cell LCR events was markedly contracted by ryanodine, suggesting a reduction in channel open time. The p[MHM/AHM](pMA) distributions were invariant across all cell types and were consistent with the model, underlying the common physical basis of their geometry. The geometric scaling of LCR events demonstrated here may help with their mechanistic characterization.
Assuntos
Cálcio/metabolismo , Modelos Biológicos , Miócitos de Músculo Liso/metabolismo , Algoritmos , Animais , Células Cultivadas , Simulação por Computador , Fundo Gástrico/citologia , Cobaias , Microscopia Confocal/métodos , Artéria Cerebral Média/citologia , Veia Porta/citologia , Coelhos , Ratos , Rianodina/metabolismo , Rianodina/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
The present study was designed to investigate the properties of spontaneous transient inward currents generated by interstitial cells (ICs) in the rabbit portal vein. Single ICs were freshly isolated from smooth muscle of the rabbit portal vein enzymetically. Using whole-cell patch clamp techniques, the spontaneous transient inward currents (STICs) were recorded at -60 mV of holding potential in freshly dispersed ICs. Both gadolinium, a non-selective cation channel inhibitor, and niflumic acid, a calcium-activated chloride channel blocker, abolished the inward currents. Replacement of external Na(+) with N-methyl-d-glucamine (NMDG(+)) also blocked the inward currents. The inward currents were abolished by caffeine, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), thapsigargin and ryanodine, but were partly inhibited by 2-aminoethoxydiphenyl borate (2-APB). W-7, a calmodulin inhibitor, increased the amplitude of the inward currents. These results suggest that non-selective cation channels are involved in the generation of the spontaneous transient inward currents recorded from ICs. The currents are regulated by intracellular calcium and calmodulin. But in the present study, the involvement of the calcium-activated chloride channels in the generation of the currents cannot be excluded.
Assuntos
Células Intersticiais de Cajal/fisiologia , Potenciais da Membrana/fisiologia , Miócitos de Músculo Liso/fisiologia , Veia Porta/fisiologia , Animais , Cálcio/metabolismo , Calmodulina/farmacologia , Células Cultivadas , Feminino , Gadolínio/farmacologia , Glutamatos/farmacologia , Células Intersticiais de Cajal/efeitos dos fármacos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Ácido Niflúmico/farmacologia , Técnicas de Patch-Clamp , Veia Porta/citologia , Coelhos , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
It is now well established that smooth muscle of the portal vein exhibits spontaneous rhythmic contraction in vitro. The present study was designed to investigate the pacemaking mechanism(s) underlying the spontaneous rhythmic contractions in the rabbit portal vein (RPV). Using whole-cell patch clamp techniques, spontaneous inward currents were recorded at -60 mV of holding potential in freshly dispersed c-Kit immunopositive interstitial cells (ICs) isolated from sections of RPV. The inward currents were abolished by caffeine, FCCP, thapsigargin, and ryanodine, but were partially inhibited by 2-APB. Both gadolinium, a non-selective cation channel inhibitor, and niflumic acid, a chloride channel blocker, inhibited the inward currents completely. Replacement of external Na(+) with NMDG(+) also blocked the inward currents. W-7, a calmodulin inhibitor, increased both the amplitude and frequency of the inward currents. Taken together, these results indicate that non-selective cationic channels are involved in the generation of spontaneous inward currents recorded from ICs. Intracellular calcium concentration and calmodulin regulate the spontaneous inward currents, which may account for spontaneous rhythmic contraction in the RPV, but a role of chloride channels may not be excluded in the present study.
Assuntos
Células Intersticiais de Cajal/fisiologia , Veia Porta/citologia , Animais , Cafeína/farmacologia , Calmodulina/farmacologia , Feminino , Gadolínio/farmacologia , Masculino , Ácido Niflúmico/farmacologia , Técnicas de Patch-Clamp , Proteínas Proto-Oncogênicas c-kit/metabolismo , Coelhos , Sorbitol/análogos & derivados , Sorbitol/farmacologia , Marcadores de Spin , Sulfonamidas/farmacologia , Tiocarbamatos/farmacologia , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
Future treatments for chronic liver disease are likely to involve manipulation of liver progenitor cells (LPCs). In the human, data characterising the regenerative response is limited and the origin of adult LPCs is unknown. However, these remain critical factors in the design of cell-based liver therapies. The developing human liver provides an ideal model to study cell lineage derivation from progenitors and to understand how foetal haematopoiesis and liver development might explain the nature of the adult LPC population. In 1st trimester human liver, portal venous endothelium (PVE) expressed adult LPC markers and markers of haematopoietic progenitor cells (HPCs) shared with haemogenic endothelium found in the embryonic dorsal aorta. Sorted PVE cells were able to generate hepatoblast-like cells co-expressing CK18 and CK19 in addition to Dlk/pref-1, E-cadherin, albumin and fibrinogen in vitro. Furthermore, PVE cells could initiate haematopoiesis. These data suggest that PVE shares phenotypical and functional similarities both with adult LPCs and embryonic haemogenic endothelium. This indicates that a temporal relationship might exist between progenitor cells in foetal liver development and adult liver regeneration, which may involve progeny of PVE.
Assuntos
Endotélio Vascular/citologia , Células Epiteliais/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Fígado/embriologia , Veia Porta/citologia , Células-Tronco/fisiologia , Biomarcadores/metabolismo , Linhagem da Célula , Ensaio de Unidades Formadoras de Colônias , Endotélio Vascular/fisiologia , Feminino , Feto/metabolismo , Imunofluorescência , Hematopoese , Células-Tronco Hematopoéticas/citologia , Humanos , Fígado/fisiologia , Fenótipo , Veia Porta/fisiologia , Gravidez , Primeiro Trimestre da Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We investigated synergism between inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) and diacylglycerol (DAG) on TRPC6-like channel activity in rabbit portal vein myocytes using single channel recording and immunoprecipitation techniques. Ins(1,4,5)P(3) at 10 microm increased 3-fold TRPC6-like activity induced by 10 microm 1-oleoyl-2-acetyl-sn-glycerol (OAG), a DAG analogue. Ins(1,4,5)P(3) had no effect on OAG-induced TRPC6 activity in mesenteric artery myocytes. Anti-TRPC6 and anti-TRPC7 antibodies blocked channel activity in portal vein but only anti-TRPC6 inhibited activity in mesenteric artery. TRPC6 and TRPC7 proteins strongly associated in portal vein but only weakly associated in mesenteric artery tissue lysates. Therefore in portal vein the conductance consists of TRPC6/C7 subunits, while OAG activates a homomeric TRPC6 channel in mesenteric artery myocytes. Wortmannin at 20 microm reduced phosphatidylinositol 4,5-bisphosphate (PIP(2)) association with TRPC6 and TRPC7, and produced a 40-fold increase in OAG-induced TRPC6/C7 activity. Anti-PIP(2) antibodies evoked TRPC6/C7 activity, which was blocked by U73122, a phospholipase C inhibitor. DiC8-PIP(2), a water-soluble PIP(2) analogue, inhibited OAG-induced TRPC6/C7 activity with an IC(50) of 0.74 microm. Ins(1,4,5)P(3) rescued OAG-induced TRPC6/C7 activity from inhibition by diC8-PIP(2) in portal vein myocytes, and this was not prevented by the Ins(1,4,5)P(3) receptor antagonist heparin. In contrast, Ins(1,4,5)P(3) did not overcome diC8-PIP(2)-induced inhibition of TRPC6 activity in mesenteric artery myocytes. 2,3,6-Tri-O-butyryl-Ins(1,4,5)P(3)/AM (6-Ins(1,4,5)P(3)), a cell-permeant analogue of Ins(1,4,5)P(3), at 10 microm increased TRPC6/C7 activity in portal vein and reduced association between TRPC7 and PIP(2), but not TRPC6 and PIP(2). In contrast, 10 microm OAG reduced association between TRPC6 and PIP(2), but not between TRPC7 and PIP(2). The present work provides the first evidence that Ins(1,4,5)P(3) modulates native TRPC channel activity through removal of the inhibitory action of PIP(2) from TRPC7 subunits.
Assuntos
Diglicerídeos/farmacologia , Inositol 1,4,5-Trifosfato/farmacologia , Miócitos de Músculo Liso/fisiologia , Fosfatidilinositol 4,5-Difosfato/fisiologia , Canais de Cátion TRPC/fisiologia , Animais , Axônios/fisiologia , Western Blotting , Eletrofisiologia , Estrenos/farmacologia , Imunoprecipitação , Ativação do Canal Iônico/efeitos dos fármacos , Artérias Mesentéricas/citologia , Artérias Mesentéricas/efeitos dos fármacos , Técnicas de Patch-Clamp , Inibidores de Fosfodiesterase/farmacologia , Veia Porta/citologia , Veia Porta/efeitos dos fármacos , Pirrolidinonas/farmacologia , Coelhos , Canais de Cátion TRPC/efeitos dos fármacosRESUMO
This study sought to probe into the mechanism of spontaneous contraction of portal vein. The morphological and electrophysiological characteristics of the freshly isolated interstitial cells (ICs) of rabbit portal vein were investigated by using immunohistochemical and conventional whole-cell patch clamp techniques. The isolated interstitial cells exhibited stellate-shaped or spindle-shaped bodies with a variable number of thin processes projecting from cell bodies, and these cells were noted to be c-Kit immunopositive. Under conventional whole-cell patch clamp configuration, the membrane potential was held at -60 mV, the spontaneous rhythmic inward currents were recorded in ICs, and the frequencies of which were similar to those of spontaneous contraction of portal vein. The inward currents were insensitive to nicardipine (an L-type calcium channel blocker) but could be abolished by gadolinium (a non-selective cation channel blocker). The results suggested that the spontaneous rhythmic inward currents recorded in freshly isolated ICs may be pacemaker currents which elicit the spontaneous contraction of portal vein.
Assuntos
Potenciais de Ação , Células Intersticiais de Cajal/fisiologia , Periodicidade , Veia Porta/fisiologia , Canais de Potencial de Receptor Transitório/metabolismo , Animais , Eletrofisiologia , Feminino , Masculino , Músculo Liso Vascular/fisiologia , Veia Porta/citologia , CoelhosRESUMO
BACKGROUND AND PURPOSE: The sarcoplasmic reticulum (SR), regulates the cytoplasmic Ca(2+) concentration ([Ca(2+)](cyto)) in vascular smooth muscle. Release from the SR is controlled by two intracellular receptor/channel complexes, the ryanodine receptor (RyR) and the inositol 1,4,5-trisphosphate receptor (IP(3)R). These receptors may be regulated by the accessory FK506-binding protein (FKBP) either directly, by binding to the channel, or indirectly via FKBP modulation of two targets, the phosphatase, calcineurin or the kinase, mammalian target of rapamycin (mTOR). EXPERIMENTAL APPROACH: Single portal vein myocytes were voltage-clamped in whole cell configuration and [Ca(2+)](cyto) measured using fluo-3. IP(3)Rs were activated by photolysis of caged IP(3) and RyRs activated by hydrostatic application of caffeine. KEY RESULTS: FK506 which displaces FKBP from each receptor (to inhibit calcineurin) increased the [Ca(2+)](cyto) rise evoked by activation of either RyR or IP(3)R. Rapamycin which displaces FKBP (to inhibit mTOR) also increased the amplitude of the caffeine-evoked, but reduced the IP(3)-evoked [Ca(2+)](cyto) rise. None of the phosphatase inhibitors, cypermethrin, okadaic acid or calcineurin inhibitory peptide, altered either caffeine- or IP(3)-evoked [Ca(2+)](cyto) release; calcineurin did not contribute to FK506-mediated potentiation of RyR- or IP(3)R-mediated Ca(2+) release. The mTOR inhibitor LY294002, like rapamycin, decreased IP(3)-evoked Ca(2+) release. CONCLUSIONS AND IMPLICATIONS: Ca(2+) release in portal vein myocytes, via RyR, was modulated directly by FKBP binding to the channel; neither calcineurin nor mTOR contributed to this regulation. However, IP(3)R-mediated Ca(2+) release, while also modulated directly by FKBP may be additionally regulated by mTOR. Rapamycin inhibition of IP(3)-mediated Ca(2+) release may be explained by mTOR inhibition.