RESUMO
OBJECTIVE: Shear stress patterns influence atherogenesis and plaque stability; low laminar shear stress (LLSS) promotes unstable plaques whereas oscillatory shear stress (OSS) induces more stable plaques. Endothelial connexin37 (Cx37) expression is also regulated by shear stress, which may contribute to localization of atherosclerotic disease. Moreover, Cx37 reduces initiation of atherosclerosis by inhibiting monocyte adhesion. The present work investigates the effect of Cx37 on the phenotype of plaques induced by LLSS or OSS. METHODS: Shear stress-modifying casts were placed around the common carotid artery of ApoE(-/-) or ApoE(-/-)Cx37(-/-) mice, and animals were placed on a high-cholesterol diet for 6 or 9 weeks. Atherosclerotic plaque size and composition were assessed by immunohistochemistry. RESULTS: Plaque size in response to OSS was increased in ApoE(-/-)Cx37(-/-) mice compared to ApoE(-/-) animals. Most plaques contained high lipid and macrophage content and a low amount of collagen. In ApoE(-/-) mice, macrophages were more prominent in LLSS than OSS plaques. This difference was reversed in ApoE(-/-)Cx37(-/-) animals, with a predominance of macrophages in OSS plaques. The increase in macrophage content in ApoE(-/-)Cx37(-/-) OSS plaques was mainly due to increased accumulation of M1 and Mox macrophage subtypes. Cx37 expression in macrophages did not affect their proliferation or their polarization in vitro. CONCLUSION: Cx37 deletion increased the size of atherosclerotic lesions in OSS regions and abrogated the development of a stable plaque phenotype under OSS in ApoE(-/-) mice. Hence, local hemodynamic factors may modify the risk for adverse atherosclerotic disease outcomes associated to a polymorphism in the human Cx37 gene.
Assuntos
Apolipoproteínas E/genética , Conexinas/genética , Placa Aterosclerótica/genética , Trifosfato de Adenosina/química , Animais , Apoptose , Aterosclerose , Adesão Celular , Diferenciação Celular , Colesterol/química , Conexinas/fisiologia , Feminino , Deleção de Genes , Hemodinâmica , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/citologia , Oscilometria , Fenótipo , Placa Aterosclerótica/metabolismo , Polimorfismo Genético , Resistência ao Cisalhamento , Proteína alfa-4 de Junções ComunicantesRESUMO
Pannexin1 (Panx1) forms ATP channels that play a critical role in the immune response by reinforcing purinergic signal amplification in the immune synapse. Platelets express Panx1 and given the importance of ATP release in platelets, we investigated Panx1 function in platelet aggregation and the potential impact of genetic polymorphisms on Panx1 channels. We show here that Panx1 forms ATP release channels in human platelets and that inhibiting Panx1 channel function with probenecid, mefloquine or specific (10)Panx1 peptides reduces collagen-induced platelet aggregation but not the response induced by arachidonic acid or ADP. These results were confirmed using Panx1-/- platelets. Natural variations have been described in the human Panx1 gene, which are predicted to induce non-conservative amino acid substitutions in its coding sequence. Healthy subjects homozygous for Panx1-400C, display enhanced platelet reactivity in response to collagen compared with those bearing the Panx1-400A allele. Conversely, the frequency of Panx1-400C homozygotes was increased among cardiovascular patients with hyper-reactive platelets compared with patients with hypo-reactive platelets. Exogenous expression of polymorphic Panx1 channels in a Panx-deficient cell line revealed increased basal and stimulated ATP release from cells transfected with Panx1-400C channels compared with Panx1-400A expressing transfectants. In conclusion, we demonstrate a specific role for Panx1 channels in the signalling pathway leading to collagen-induced platelet aggregation. Our study further identifies for the first time an association between a Panx1-400A>C genetic polymorphism and collagen-induced platelet reactivity. The Panx1-400C variant encodes for a gain-of-function channel that may adversely affect atherothrombosis by specifically enhancing collagen-induced ATP release and platelet aggregation.
Assuntos
Colágeno/farmacologia , Conexinas/genética , Proteínas do Tecido Nervoso/genética , Agregação Plaquetária/fisiologia , Polimorfismo de Nucleotídeo Único , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/metabolismo , Adolescente , Adulto , Alelos , Substituição de Aminoácidos , Animais , Ácido Araquidônico/farmacologia , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/genética , Conexinas/deficiência , Conexinas/fisiologia , Frequência do Gene , Genótipo , Humanos , Masculino , Mefloquina/farmacologia , Camundongos Knockout , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/fisiologia , Fragmentos de Peptídeos/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Probenecid/farmacologia , Antagonistas do Receptor Purinérgico P2X/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Transfecção , Adulto JovemRESUMO
Ubiquitous reduction of the gap junction protein Connexin43 (Cx43) in mice provides beneficial effects on progression and composition of atherosclerotic lesions. Cx43 is expressed in multiple atheroma-associated cells but its function in each cell type is not known. To examine specifically the role of Cx43 in immune cells, we have lethally irradiated low-density lipoprotein receptor-deficient mice and reconstituted with Cx43+/+, Cx43+/- or Cx43-/- haematopoietic fetal liver cells. Progression of atherosclerosis was significantly lower in aortic roots of Cx43+/- chimeras compared with Cx43+/+ and Cx43-/- chimeras, and their plaques contained significantly less neutrophils. The relative proportion of circulating leukocytes was similar between the three groups. Interestingly, the chemoattraction of neutrophils, which did not express Cx43, was reduced in response to supernatant secreted by Cx43+/- macrophages in comparison with the ones of Cx43+/+ and Cx43-/- macrophages. Cx43+/- macrophages did not differ from Cx43+/+ and Cx43-/- macrophages in terms of M1/M2 polarisation but show modified gene expression for a variety chemokines and complement components. In conclusion, titration of Cx43 expression in bone marrow-derived macrophages reduces atherosclerotic plaque formation and chemoattraction of neutrophils to the lesions.
Assuntos
Aorta/metabolismo , Doenças da Aorta/prevenção & controle , Aterosclerose/prevenção & controle , Conexina 43/metabolismo , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Animais , Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/imunologia , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Células Cultivadas , Quimiotaxia de Leucócito , Técnicas de Cocultura , Conexina 43/deficiência , Conexina 43/genética , Modelos Animais de Doenças , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infiltração de Neutrófilos , Fenótipo , Placa Aterosclerótica , Interferência de RNA , Receptores de LDL/deficiência , Receptores de LDL/genética , Transfecção , Irradiação Corporal TotalRESUMO
The recovery of an intact epithelium following injury is critical for restoration of lung homeostasis, a process that may be altered in cystic fibrosis (CF). In response to injury, progenitor cells in the undamaged areas migrate, proliferate and re-differentiate to regenerate an intact airway epithelium. The mechanisms regulating this regenerative response are, however, not well understood. In a model of circular wound injury of well-differentiated human airway epithelial cell (HAEC) cultures, we identified the gap junction protein Cx26 as an important regulator of cell proliferation. We report that induction of Cx26 in repairing HAECs is associated with cell proliferation. We also show that Cx26 is expressed in a population of CK14-positive basal-like cells. Cx26 silencing in immortalized cell lines using siRNA and in primary HAECs using lentiviral-transduced shRNA enhanced Ki67-labeling index and Ki67 mRNA, indicating that Cx26 acts a negative regulator of HAEC proliferation. Cx26 silencing also markedly decreased the transcription of KLF4 in immortalized HAECs. We further show that CF HAECs exhibited deregulated expression of KLF4, Ki67 and Cx26 as well enhanced rate of wound closure in the early response to injury. These results point to an altered repair process of CF HAECs characterized by rapid but desynchronized initiation of HAEC activation and proliferation.
Assuntos
Brônquios/metabolismo , Brônquios/patologia , Conexinas/metabolismo , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Conexina 26 , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismoRESUMO
BACKGROUND: Endothelial dysfunction is the initiating event of atherosclerosis. The expression of connexin40 (Cx40), an endothelial gap junction protein, is decreased during atherogenesis. In the present report, we sought to determine whether Cx40 contributes to the development of the disease. METHODS AND RESULTS: Mice with ubiquitous deletion of Cx40 are hypertensive, a risk factor for atherosclerosis. Consequently, we generated atherosclerosis-susceptible mice with endothelial-specific deletion of Cx40 (Cx40del mice). Cx40del mice were indeed not hypertensive. The progression of atherosclerosis was increased in Cx40del mice after 5 and 10 weeks of a high-cholesterol diet, and spontaneous lesions were observed in the aortic sinuses of young mice without such a diet. These lesions showed monocyte infiltration into the intima, increased expression of vascular cell adhesion molecule-1, and decreased expression of the ecto-enzyme CD73 in the endothelium. The proinflammatory phenotype of Cx40del mice was confirmed in another model of induced leukocyte recruitment from the lung microcirculation. Endothelial CD73 is known to induce antiadhesion signaling via the production of adenosine. We found that reducing Cx40 expression in vitro with small interfering RNA or antisense decreased CD73 expression and activity and increased leukocyte adhesion to mouse endothelial cells. These effects were reversed by an adenosine receptor agonist. CONCLUSIONS: Cx40-mediated gap junctional communication contributes to a quiescent nonactivated endothelium by propagating adenosine-evoked antiinflammatory signals between endothelial cells. Alteration in this mechanism by targeting Cx40 promotes leukocyte adhesion to the endothelium, thus accelerating atherosclerosis.
Assuntos
5'-Nucleotidase/metabolismo , Aterosclerose/fisiopatologia , Conexinas/genética , Células Endoteliais/patologia , Vasculite/fisiopatologia , Animais , Aterosclerose/imunologia , Aterosclerose/patologia , Adesão Celular/imunologia , Células Cultivadas , Conexinas/metabolismo , Células Endoteliais/metabolismo , Junções Comunicantes/metabolismo , Proteínas de Fluorescência Verde/genética , Camundongos , Camundongos Transgênicos , Monócitos/metabolismo , Monócitos/patologia , RNA Interferente Pequeno , Transdução de Sinais/imunologia , Vasculite/imunologia , Vasculite/patologia , Proteína alfa-5 de Junções ComunicantesAssuntos
Anti-Inflamatórios/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Hipolipemiantes/farmacologia , Ativação de Neutrófilo/efeitos dos fármacos , Animais , Arteriosclerose/imunologia , Arteriosclerose/prevenção & controle , HumanosRESUMO
Wounding of endothelial cells is associated with altered direct intercellular communication. To determine whether gap junctional communication participates to the wound repair process, we have compared connexin (Cx) expression, cell-to-cell coupling and kinetics of wound repair in monolayer cultures of PymT-transformed mouse endothelial cells (clone bEnd.3) and in bEnd.3 cells expressing different dominant negative Cx inhibitors. In parental bEnd.3 cells, mechanical wounding increased expression of Cx43 and decreased expression of Cx37 at the site of injury, whereas Cx40 expression was unaffected. These wound-induced changes in Cx expression were associated with functional changes in cell-to-cell coupling, as assessed with different fluorescent tracers. Stable transfection with cDNAs encoding for the chimeric connexin 3243H7 or the fusion protein Cx43-betaGal resulted in perturbed gap junctional communication between bEnd.3 cells under both basal and wounded conditions. The time required for complete repair of a defined wound within a confluent monolayer was increased by ~50% in cells expressing the dominant negative Cx inhibitors, whereas other cell properties, such as proliferation rate, migration of single cells, cyst formation and extracellular proteolytic activity, were unaltered. These findings demonstrate that proper Cx expression is required for coordinated migration during repair of an endothelial wound.
Assuntos
Conexina 43/metabolismo , Conexinas/metabolismo , Endotélio Vascular/lesões , Cicatrização , Animais , Comunicação Celular , Linhagem Celular , Conexina 43/genética , Conexinas/genética , Endotélio Vascular/citologia , Expressão Gênica , Células HeLa , Humanos , Camundongos , Proteína alfa-4 de Junções ComunicantesRESUMO
1. The aim of this study was to inhibit specifically one type of gap junction channel in cells expressing multiple connexins (Cx) using synthetic oligopeptides. 2. A7r5 cells (an aortic smooth muscle cell line expressing Cx40 and Cx43) were incubated overnight with synthetic oligopeptides (P180-195) corresponding to a segment of the second extracellular loop of Cx43. This segment is different in sequence from the corresponding location in Cx40. 3. P180-195 (500 microM) decreased cell-to-cell coupling as assessed by dye coupling and dual whole-cell voltage clamp. The decrease in permeability and junctional conductance was caused by selective inhibition of Cx43 gap junction channels. In contrast, overnight incubation of A7r5 cells with oligopeptides corresponding to a segment of the intracellular cytoplasmic tail of Cx43 was without effect. 4. These results indicate that oligopeptides P180-195 may interact with the extracellular domain of the Cx43 protein, thereby possibly mimicking connexin-connexin binding. This apparently inhibits Cx43 channel activity without disturbing the activity of Cx40 channels. 5. Experiments with oligopeptides corresponding to the equivalent part of the second extracellular loop of Cx40 (P177-192) pointed towards a selective inhibition of Cx40 channel activity. 6. Competition assays using synthetic oligopeptides may help to resolve the regulatory properties of gap junction channels in primary cells expressing multiple Cx.
Assuntos
Junções Comunicantes/efeitos dos fármacos , Oligopeptídeos/farmacologia , Animais , Linhagem Celular , Conexinas/biossíntese , Eletrofisiologia , Espaço Extracelular/metabolismo , Imunofluorescência , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Oligopeptídeos/síntese química , Técnicas de Patch-Clamp , RatosRESUMO
OBJECTIVE: Primary cultures of neonatal rat ventricular myocytes have become a widely used model to examine a variety of functional, physiological and biochemical cardiac properties. In the adult rat, connexin43 (Cx43) is the major gap junction protein present in the working myocardium. In situ hybridization studies on developing rats, however, showed that Cx40 mRNA displays a dynamic and heterogeneous pattern of expression in the ventricular myocardium around birth. The present studies were performed to examine the expression pattern of the Cx40 protein in neonatal rat heart, and to examine the connexins present in cultures of ventricular myocytes obtained from those hearts. METHODS: Cryosections were made of hearts of 1-day-old Wistar rats. Cultures of ventricular myocytes obtained from these hearts by enzymatic dissociation were seeded at various densities (to obtain > 75, approximately 50%, and < 25% confluency) and cultured for 24, 48 or 96 h. Cx40 and Cx43 were detected by immunofluorescence and immunoblotting. RESULTS: Immunohistochemical stainings confirmed that gap junctions in the atrium and His-Purkinje system were composed of at least Cx43 and Cx40. From the subendocardium towards the subepicardium Cx40 expression gradually decreased, resulting in the sole expression of Cx43 in the subepicardial part of the ventricular wall. In ventricular myocytes cultured at high density (> 75% confluency) Cx43 and Cx40 immunoreactivity could be detected. In contrast to Cx43 immunolabeling which showed a homogeneous distribution pattern, Cx40 staining was heterogeneous, i.e. in some clusters of cells abundant labeling was present whereas in others no Cx40 staining could be detected. The pattern of Cx43 immunoreactivity was not altered by the culture density. In contrast, in isolated ventricular myocytes cultured at low density (< 25% confluency) the relative number of cell-cell interfaces that were Cx40-immunopositive decreased as compared to high density cultures (35 vs. 70%). Western blots did not reveal significant differences in the level of Cx40 and Cx43 expression at different culture densities. CONCLUSIONS: These results show that cultured ventricular myocytes retained typical features of the native neonatal rat ventricular myocardium with regard to their composition of gap junctions. This implicates that these cultures may serve as a good model for studying short-term and long-term regulation of cardiac gap junction channel expression and function.
Assuntos
Animais Recém-Nascidos/metabolismo , Conexina 43/análise , Junções Comunicantes/metabolismo , Miocárdio/metabolismo , Animais , Western Blotting , Células Cultivadas , Conexinas/análise , Microscopia de Fluorescência , Modelos Biológicos , Ratos , Ratos Wistar , Proteína alfa-5 de Junções ComunicantesRESUMO
For effective cardiac output, it is essential that electrical excitation spread rapidly throughout the atria and ventricles. This is effected by electrical coupling through gap junction channels at contact sites between myocytes. These channels form a low-resistance pathway between adjacent myocytes and consist of connexin proteins. The connexin family is a large multigene family, and the channels formed by different members of this family have distinct electrical and regulatory properties. We have studied gap junction channels between adult rabbit atrial and ventricular myocytes using immunocytochemical and electrophysiological methods. Gap junctions of ventricular myocytes were immunoreactive to antibodies directed against connexin43 (Cx43) and Cx45, but not to antibodies against Cx37 or Cx40. Gap junctions between atrial myocytes showed immunostaining with anti-Cx40, -Cx43, and -Cx45 antibodies, but not with anti-Cx37 antibody. Endocardial and endothelial tissue were labeled with both Cx37 and Cx40 antibodies. The conductance of rabbit myocardial gap junctions was measured using the double whole-cell voltage-clamp method. The average macroscopic junctional conductance, corrected for series resistance, of atrial and ventricular cell pairs did not differ significantly (169+/-146 and 175+/-147 nS, respectively), and both were at most only slightly sensitive to the applied transjunctional potential difference. The difference in connexin expression between atrial and ventricular myocytes was reflected in the distribution of single gap junction channel conductances. A single population of unitary channel conductances with an average of 100 pS was observed between ventricular myocyte pairs. In addition to this population, a population with an average conductance of 185 pS was present between atrial myocyte pairs. The observed difference in connexin expression between atrial and ventricular myocardium may enable differential regulation of conduction in these tissues.
Assuntos
Função Atrial , Conexinas/fisiologia , Junções Comunicantes/fisiologia , Miocárdio/química , Função Ventricular , Potenciais de Ação , Animais , Conexina 43/análise , Conexina 43/imunologia , Conexina 43/fisiologia , Conexinas/análise , Conexinas/imunologia , Eletrofisiologia , Junções Comunicantes/química , Átrios do Coração/química , Átrios do Coração/citologia , Ventrículos do Coração/química , Ventrículos do Coração/citologia , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Miocárdio/citologia , Coelhos , Proteína alfa-5 de Junções Comunicantes , Proteína alfa-4 de Junções ComunicantesRESUMO
Short term (15 min) effects of activators of protein kinase A (PKA), PKC and PKG on cardiac macroscopic (g(j)) and single channel (gamma j) gap junctional conductances were studied in pairs of neonatal rat cardiomyocytes. Under dual whole-cell voltage-clamp, PKC activation by 100 nM TPA increased g(j) by 16 +/- 2% (mean +/- S.E.M, n = 9), 1.5 mM of the PKG activator 8-bromo-cGMP (8Br-cGMP) decreased g(j) by 26 +/- 2% (n = 4), whereas 1.5 mM of the PKA activator 8Br-cAMP did not affect g(j) (1 +/- 5%, n = 11). Single cardiac gap junction channel events, resolved in the presence of heptanol, indicated two gamma j sizes of 20 pS and 40-45 pS. Under control conditions, the larger events were most frequently observed. Whereas 8Br-cAMP did not change this distribution, TPA or 8Br-cGMP shifted the gamma j distribution to the lower sizes. Diffusion of 6-carboxyfluorescein (6-CF), a gap junction permeant tracer, from the injected cell to neighboring cells was studied on small clusters of neonatal rat cardiomyocytes. Under control conditions, 6-CF labeled 8.4 +/- 0.4 cells (mean +/- S.E.M, n = 31). Whereas 8Br-cAMP did not change the extent of dye transfer (8.1 +/- 0.5 cells, n = 10), TPA restricted the diffusion of 6-CF to 2.2 +/- 0.2 cells (n = 30) and 8Br-cGMP to 3.5 +/- 0.3 cells (n = 10). This suggests that permeability and single channel conductance of Cx43 gap junction channels are parallel related. Altogether, these results point to the differential modulation of electrical and metabolic coupling of cardiac cells by various phosphorylating conditions.
Assuntos
Junções Comunicantes/fisiologia , Coração/fisiologia , Miocárdio/enzimologia , Proteínas Quinases/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , Conexina 43/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Condutividade Elétrica , Junções Comunicantes/efeitos dos fármacos , Coração/efeitos dos fármacos , Cinética , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Fosforilação , Proteína Quinase C/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacosRESUMO
Studies on physiological modulation of intercellular communication mediated by protein kinases are often complicated by the fact that cells express multiple gap junction proteins (connexins; Cx). Changes in cell coupling can be masked by simultaneous opposite regulation of the gap junction channel types expressed. We have examined the effects of activators and inhibitors of protein kinase A (PKA), PKC, and PKG on permeability and single channel conductance of gap junction channels composed of Cx45, Cx43, or Cx26 subunits. To allow direct comparison between these Cx, SKHep1 cells, which endogenously express Cx45, were stably transfected with cDNAs coding for Cx43 or Cx26. Under control conditions, the distinct types of gap junction channels could be distinguished on the basis of their permeability and single channel properties. Under various phosphorylating conditions, these channels behaved differently. Whereas agonists/antagonist of PKA did not affect permeability and conductance of all gap junction channels, variable changes were observed under PKC stimulation. Cx45 channels exhibited an additional conductance state, the detection of the smaller conductance states of Cx43 channels was favored, and Cx26 channels were less often observed. In contrast to the other kinases, agonists/antagonist of PKG affected permeability and conductance of Cx43 gap junction channels only. Taken together, these results show that distinct types of gap junction channels are differentially regulated by similar phosphorylating conditions. This differential regulation may be of physiological importance during modulation of cell-to-cell communication of more complex cell systems.
Assuntos
Comunicação Celular , Conexinas/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Junções Comunicantes/fisiologia , Proteína Quinase C/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Sequência de Bases , Carcinoma Hepatocelular , Conexina 26 , Conexinas/biossíntese , Proteínas Quinases Dependentes de AMP Cíclico/biossíntese , Proteínas Quinases Dependentes de AMP Cíclico/isolamento & purificação , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Proteínas Quinases Dependentes de GMP Cíclico/biossíntese , Proteínas Quinases Dependentes de GMP Cíclico/isolamento & purificação , Primers do DNA , Inibidores Enzimáticos/farmacologia , Expressão Gênica , Homeostase , Humanos , Neoplasias Hepáticas , Potenciais da Membrana/fisiologia , Dados de Sequência Molecular , Fosforilação , Reação em Cadeia da Polimerase , Proteína Quinase C/biossíntese , Proteína Quinase C/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Transfecção , Células Tumorais CultivadasRESUMO
Short-term (10 min) effects of 100 nM 12-O-tetradecanoylphorbol-13-acetate (TPA), the protein kinase C (PKC) activator, on cardiac macroscopic (gj) and single channel (gamma j) gap junctional conductances were studied in pairs of neonatal rat cardiomyocytes. Under dual whole-cell (WC) or perforated patch (PP) voltage-clamp, gj increased by 15.5 +/- 7.2% (mean +/- SD, n = 9) and by 46.3 +/- 17.0% (n = 5), respectively. The latter difference is not related to intracellular calcium concentration, because raising the Ca2+ concentration in the electrode solution did not change the TPA-induced increase in gj observed under WC conditions. The inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate (alpha PDD), did not affect gj. Single cardiac gap junction channel events, resolved in the presence of heptanol, indicated two gamma j sizes of 20 and 40-45 pS. Under control conditions, the larger events were most frequently observed. Whereas alpha PDD did not change this distribution, TPA shifted the gamma j distribution to the lower sizes. Diffusion of Lucifer Yellow (LY) and 6-carboxyfluorescein (6-CF), gap junction permeant tracers, was studied on small clusters of cardiomyocytes. Under control conditions, LY labeled 19.4 +/- 7.2 cells (mean +/- SD, n = 18) and 6-CF labeled 8.4 +/- 2.2 cells (n = 20). Whereas alpha PDD did not change the extent of dye transfer, TPA restricted the diffusion of LY to 2.8 +/- 1.3 cells (n = 11) and of 6-CF to 2.4 +/- 1.4 cells (n = 20). This suggests that permeability and single channel conductance of connexin 43 channels are parallely related. Altogether, these results point to the opposite modulation of electrical and metabolic coupling of cardiac cells evoked by TPA.
Assuntos
Permeabilidade da Membrana Celular/fisiologia , Junções Comunicantes/fisiologia , Coração/efeitos dos fármacos , Miocárdio/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Animais , Animais Recém-Nascidos , Comunicação Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , Condutividade Elétrica , Ativação Enzimática , Imunofluorescência , Junções Comunicantes/efeitos dos fármacos , Coração/fisiologia , Microscopia Confocal , Miocárdio/citologia , Técnicas de Patch-Clamp , Proteína Quinase C/metabolismo , Ratos , Ratos WistarRESUMO
The effects of 8-bromoguanosine 3':5'-cyclic monophosphate (8Br-cGMP), a membrane-permeant activator of protein kinase G (PKG), were studied on rat and human connexin43 (Cx43), the most abundant gap junction protein in mammalian heart, which were exogenously expressed in SKHep1 cells. Under dual whole-cell voltage-clamp conditions, 8Br-cGMP decreased gap junctional conductance (gj) in rat Cx43-transfected cells by 24.0 +/- 3.7% (mean +/- SEM, n = 5), whereas gj was not affected in human Cx43-transfected cells by the same treatment. The relaxation of gj in response to steps in transjunctional voltage observed in rat Cx43 transfectants was best fitted with three exponentials. Time constants and amplitudes of the decay phases changed in the presence of 8Br-cGMP. Single rat and human Cx43 gap junction channels were resolved in the presence of halothane. Under control conditions, three single-channel conductance states (gammaj) of about 20, 40-45 and 70 pS were detected, the events of the intermediate size being most frequently observed. In the presence of 8Br-cGMP, the gammaj distribution shifted to the lower size in rat Cx43 but not in human Cx43 transfectants. Immunoblot analyses of Cx43 in subconfluent cultures of rat Cx43 or human Cx43 transfectants showed that 8Br-cGMP did not induce changes in the electrophoretic mobility of Cx43 in either species. However, the basal incorporation of [32P] into rat Cx43 was significantly altered by 8Br-cGMP, whereas this incorporation of [32P] into human Cx43 was not affected. We conclude that 8Br-cGMP modulates phosphorylation of rat Cx43 in SKHep1 cells, but not of human Cx43. This cGMP-dependent phosphorylation of rat Cx43 is associated with a decreased gj, which results from both an increase in the relative frequency of the lowest conductance state and a change in the kinetics of these channels.
Assuntos
GMP Cíclico/fisiologia , Junções Comunicantes/metabolismo , Canais Iônicos/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Aminoácidos/metabolismo , Animais , Western Blotting , Carcinoma Hepatocelular/metabolismo , Linhagem Celular , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , DNA Complementar/biossíntese , Proteína GAP-43 , Humanos , Neoplasias Hepáticas/metabolismo , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp , Fosforilação , Testes de Precipitina , Proteínas Quinases/metabolismo , Ratos , TransfecçãoRESUMO
To assess whether alterations in membrane fluidity of neonatal rat heart cells modulate gap junctional conductance (gj), we compared the effects of 2 mM 1-heptanol and 20 microM 2-(methoxy-ethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)-octanoate (A2C) in a combined fluorescence anisotropy and electrophysiological study. Both substances decreased fluorescence steady-state anisotropy (rss), as assessed with the fluorescent probe 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) by 9.6 +/- 1.1% (mean +/- SEM, n = 5) and 9.8 +/- 0.6% (n = 5), respectively, i.e., both substances increased bulk membrane fluidity. Double whole-cell voltage-clamp experiments showed that 2 mM heptanol uncoupled cell pairs completely (n = 6), whereas 20 microM A2C, which increased bulk membrane fluidity to the same extent, did not affect coupling at all (n = 5). Since gap junction channels are embedded in relatively cholesterol-rich domains of the membrane, we specifically assessed the fluidity of the cholesterol-rich domains with dehydroergosterol (DHE). Using DHE, heptanol increased rss by 14.9 +/- 3.0% (n = 5), i.e., decreased cholesterol domain fluidity, whereas A2C had no effect on rss (-0.4 +/- 6.7%, n = 5). Following an increase of cellular "cholesterol" content (by loading the cells with DHE), 2 mM heptanol did not uncouple cell pairs completely: gj decreased by 80 +/- 20% (range 41-95%, n = 5). The decrease in gj was most probably due to a decrease in the open probability of the gap junction channels, because the unitary conductances of the channels were not changed nor was the number of channels comprising the gap junction. The sensitivity of nonjunctional membrane channels to heptanol was unaltered in cholesterol-enriched myocytes. These results indicate that the fluidity of cholesterol-rich domains is of importance to gap junctional coupling, and that heptanol decreases gj by decreasing the fluidity of cholesterol-rich domains, rather than by increasing the bulk membrane fluidity.
Assuntos
Álcoois/farmacologia , Colesterol/fisiologia , Junções Comunicantes/fisiologia , Coração/fisiologia , Fluidez de Membrana/efeitos dos fármacos , Lipídeos de Membrana/fisiologia , Animais , Animais Recém-Nascidos , Condutividade Elétrica/efeitos dos fármacos , Polarização de Fluorescência , Junções Comunicantes/efeitos dos fármacos , Heptanol , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/fisiologia , Miocárdio/química , Miocárdio/citologia , Ratos , Ratos Wistar , Sarcolema/efeitos dos fármacos , Sarcolema/fisiologiaRESUMO
The effects of an increase in intracellular cyclic GMP (cGMP)-concentration on gap junctional current (Ij) were studied in cultured neonatal rat heart cells using both the whole-cell and perforated patch voltage-clamp method. In whole-cell measurements, exposure to 8-bromo-cGMP or carbachol reduced Ij. With the perforated patch technique, on the other hand, Ij was not affected by either 8-bromo-cGMP or carbachol. Addition of alkaline phosphatase prevented the carbachol-induced decrease in Ij in whole-cell measurements. Reduction of Ij in well-coupled cell pairs by application of heptanol allowed us to study the effects of these substances on the single gap junction channel level. We found that cGMP-treatment shifts the single channel conductance (gamma j) from 43 to 21 pS in whole-cell measurements and that intracellular addition of phosphatase prevents this shift. In contrast, intracellular phosphatase-treatment itself shifts gamma j to 70 pS. Our results indicate that Cx43-gap junction channels may exhibit three conductance levels (21 pS, 40-45 pS and 70 pS), depending on the phosphorylation state of the protein.
Assuntos
Junções Intercelulares/fisiologia , Canais Iônicos/fisiologia , Miocárdio/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Fosfatase Alcalina/farmacologia , Animais , Animais Recém-Nascidos , Carbacol/farmacologia , Condutividade Elétrica , Junções Intercelulares/metabolismo , Canais Iônicos/metabolismo , Miocárdio/citologia , Fosforilação , RatosRESUMO
The influence of heptanol on gap junctional and non-junctional membrane currents was studied in cultured neonatal rat heart cells using both the whole cell and perforated patch voltage-clamp method. With both methods, exposure to heptanol produced a dose-dependent decrease in the junctional current (dissociation constant = 0.54 and 1.20 mM for whole cell and perforated patch experiments, respectively). Heptanol-induced uncoupling was reversible. In the same concentration range, heptanol reduced all nonjunctional membrane ionic currents examined. This suggests that heptanol does not act specifically on gap junction channels but rather on the structure of the lipid membrane. This hypothesis is strengthened by the observation that in monolayer cultures of neonatal rat heart cells fluorescence steady-state anisotropy decreased proportional with increasing the heptanol concentration in the bath. Single-channel conductances (gamma j) were identical with both recording methods (21 and 40-45 pS); heptanol did not alter gamma j. Under conditions of reduced junctional coupling induced by heptanol, junctional conductance (gj) displayed voltage sensitivity at values of gj at which no voltage sensitivity could be observed under control conditions. These results suggest that heptanol-dependent uncoupling was caused by a decrease in open probability of the gap junction channels.