Browsing by Author "Eugenín, EA"

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    Gap junctional communication coordinates vasopressin-induced glycogenolysis in rat hepatocytes
    (1998) Eugenín, EA; González, H; Sáez, CG; Sáez, JC
    Because hepatocytes communicate via gap junctions, it has been proposed that Ca2+ waves propagate through this pathway and in the process activate Ca2+-dependent cellular responses. We tested this hypothesis by measuring vasopressin-induced glycogenolysis in shortterm cultures of rat hepatocytes. A 15-min vasopressin (10(-8) M) stimulation induced a reduction of glycogen content that reached a maximum 1-3 h later. Gap junction blockers, octanol or 18 alpha-glycyrrhetinic acid, reduced the effect by 70%. The glycogenolytic response induced by Ca2+ ionophore 8-bromo-A-21387, which acts on each hepatocyte, was not affected by gap junction blockers. Moreover, the vasopressin-induced glycogenolysis was lower (70%) in dispersed than in reaggregated hepatocytes and in dispersed hepatocytes was not affected by gap junction blockers. In hepatocytes reaggregated in the presence of a synthetic peptide homologous to a domain of the extracellular loop I of the main hepatocyte gap junctional protein, vasopressin-induced glycogenolysis and incidence of dye coupling were drastically reduced. Moreover, gap junctional communication was detected between reaggregated cells, suggesting that hepatocytes with different vasopressin receptor densities become coupled to each other. The vasopressin-induced effect was not affected by suramin, ruling out ATP as a paracrine mediator. We propose that gap junctions allow for a coordinated vasopressin-induced glycogenolytic response despite the heterogeneity among hepatocytes.
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    TNF-α plus IFN-γ induce connexin43 expression and formation of gap junctions between human monocytes/macrophages that enhance physiological responses
    (2003) Eugenín, EA; Brañes, MC; Berman, JW; Sáez, JC
    In this work, the effects of bacterial LPS, TNF-alpha, and IFN-gamma on gap junctional communication (dye coupling) and on the expression of connexin43 (immunofluorescence immunoblotting, and RT-PCR) in monocytes/macrophages were studied. Freshly isolated human monocytes plated at high density and treated either with LPS plus IFN-gamma or TNF-alpha plus IFN-gamma became transiently dye coupled (Lucifer yellow) within 24 h. Cells treated with LPS, TNF-alpha, or IFN-gamma alone remained dye uncoupled. In dye-coupled cells, the spread of Lucifer yellow to neighboring cells was reversibly blocked with 18 alpha-glycyrrhetinic acid, a gap junction blocker, but it was unaffected by oxidized ATP or probenecid, which block ionotropic ATP-activated channels and organic anion transporters, respectively. Abs against TNF-alpha significantly reduced the LPS plus IFN-gamma-induced increase in dye coupling. In dye-coupled monocytes/macrophages, but not in control cells, both connexin43 protein and mRNA were detected, and their levels were higher in cells with an elevated incidence of dye coupling. In dye-coupled cells, the localization of connexin43 immunoreactivity was diffuse at perinuclear regions and thin cell processes. The addition of 18-alpha-glycyrrhetinic acid induced a profound reduction of monocyte/macrophage transmigration across a blood brain barrier model. It also induced a significant reduction in the secretion of metalloproteinase-2 in cells treated with TNF-alpha plus IFN-gamma. We propose that some monocyte/macrophage responses are coordinated by connexin-formed membrane channels expressed transiently at inflammatory sites in which these cells form aggregates.