RESUMO
The polarity of cell-surface membrane movements and their regulation by adrenal steroid hormones (10(-6) M aldosterone) and vasopressin or vasotocin were studied in A6 cells. This cell line is derived from the Xenopus laevis distal nephron and displays regulated Na+ reabsorption but is devoid of regulated water transport. Apical and basolateral membrane movements and their hormonal regulation were characterized by measuring the uptake of the fluid phase marker horseradish peroxidase (HRP) and the secretion of proteins on both sides of cell monolayers cultured on filters. The intracellular accumulation of HRP was visualized by electron microscopy and quantified by the measure of cell-associated peroxidase activity. The rate of intracellular HRP accumulation corresponded to 0.01 nl/minute/filter (4.7 cm2) from the apical side and was 20-32 times faster from the basolateral side. In contrast, the level of protein secretion was 3.5 times higher apically than basolaterally. Among the secreted proteins some were found to be secreted essentially apically, and others basolaterally. Vasotocin increased apical endocytosis (1.88-fold) and apical protein secretion (1.49-fold) in cells pretreated with aldosterone. Basolaterally, only the endocytosis was increased, and to a smaller extent (1.36-fold). These effects of vasotocin depended on aldosterone pretreatment and could be mimicked with forskolin and 8-bromoadenosine 3':5'-cyclic monophosphate (BrcAMP). Measurements of intracellular cAMP levels showed that there was a rankorder correlation between the induced level of intracellular cAMP and that of apical endocytosis. This study shows that vasotocin has a polarized stimulatory action on apical endocytosis and protein secretion in A6 cells, and that the mediation of this action by cAMP is aldosterone dependent.
Assuntos
Aldosterona/farmacologia , Néfrons/citologia , Néfrons/fisiologia , Vasopressinas/farmacologia , Vasotocina/farmacologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Colforsina/farmacologia , AMP Cíclico/análise , AMP Cíclico/metabolismo , AMP Cíclico/fisiologia , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Células Epiteliais , Epitélio/fisiologia , Epitélio/ultraestrutura , Peroxidase do Rábano Silvestre , Fluidez de Membrana/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Microscopia Eletrônica , Néfrons/ultraestrutura , Sistemas do Segundo Mensageiro/fisiologia , Sódio/farmacocinética , Xenopus laevisRESUMO
Antidiuretic hormone (ADH; 2.5 x 10(-8) M vasotocin) produces a stimulation of apical fluid phase endocytosis, protein secretion and NaCl reabsorption in Xenopus laevis A6 distal nephron cell epithelia pretreated with aldosterone (10(-6) M). The increase of NaCl transport is mediated by a sequential opening of apical Cl and Na conductances. The aim of this study was to characterize the actin and tubulin cytoskeleton of A6 cells and to assess the impact of its disruption on baseline and ADH-induced apical vesicular membrane movements and ion transport to test for possible functional links. The microfilament (MF) and microtubule (MT) networks and their disruption were visualized by confocal laser microscopy. Conditions of depolimerization were selected, by cytochalasin D or cold and nocodazole, respectively. MF disruption produced an increase in baseline apical protein secretion (exocytic movements) (plus 18%) and a decrease of its induction by ADH (minus 35%). MF disruption also increased baseline horseradish peroxidase uptake (endocytic movements) (plus 21%), however, without affecting its ADH-induced increase. In the case of MT disruption, the ADH-induced stimulation of both protein secretion and fluid phase endocytosis was decreased by 70 and 44%, respectively. At the ion transport level, MF and MT disruption only insignificantly affected the ADH-induced Cl conductance, while they decreased the ADH-induced stimulation of Na transport (amiloride-sensitive short-circuit current and conductance) by a factor of 2 to 4. In conclusion, both MT and MF disruption decrease ADH-induced apical protein secretion and Na conductance, while the ADH-induced apical Cl conductance is not significantly affected. Taken together the data support the hypothesis that the modulation of Na channel expression by apical vesicular membrane movements plays a role in Na transport expression and its regulation by ADH.
Assuntos
Actinas/análise , Citoesqueleto/fisiologia , Endocitose/efeitos dos fármacos , Exocitose/efeitos dos fármacos , Túbulos Renais Distais/efeitos dos fármacos , Canais de Sódio/fisiologia , Cloreto de Sódio/metabolismo , Tubulina (Proteína)/análise , Vasopressinas/farmacologia , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/fisiologia , Citoesqueleto de Actina/ultraestrutura , Aldosterona/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Biopolímeros , Linhagem Celular , Polaridade Celular , Canais de Cloreto/efeitos dos fármacos , Canais de Cloreto/metabolismo , Temperatura Baixa , AMP Cíclico/fisiologia , Citocalasina D/farmacologia , Citoesqueleto/efeitos dos fármacos , Canais Epiteliais de Sódio , Epitélio/efeitos dos fármacos , Epitélio/fisiologia , Epitélio/ultraestrutura , Túbulos Renais Distais/fisiologia , Túbulos Renais Distais/ultraestrutura , Potenciais da Membrana/efeitos dos fármacos , Microscopia Eletrônica de Varredura , Microtúbulos/efeitos dos fármacos , Microtúbulos/fisiologia , Microtúbulos/ultraestrutura , Nocodazol/farmacologia , Proteínas/metabolismo , Canais de Sódio/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/metabolismo , Xenopus laevisRESUMO
Non-activated follicular lymphoma (FL) cells do not function as effective antigen-presenting cells in vivo. CD40 activation of FL cells up-regulates critical adhesion and co-stimulatory molecules, thereby inducing lymphoma-specific cytotoxic T cells in vitro. However, other evidence suggests that CD70 is another important co-stimulatory molecule involved in antigen dependent T-cell activation. Here, we showed that freshly isolated FL cells from eight diagnostic biopsies expressed intermediate to high levels of CD27, whereas only insignificant levels of CD70 were detected on their cell surface. Together with the low to intermediate expression of B7, these findings help to explain the poor antigen-presenting capacity of non-activated FL cells. Activation of FL cells by CD27 and CD40 induced a significantly higher alloantigen T-cell response than CD40 alone, whereas CD27 activation induced only a mostly insignificant T-cell proliferation. Both CD40 and CD27 + CD40 activation resulted in a high up-regulation of CD70 and B7 molecules, whereas CD27 activation up-regulated CD70 but not B7 expression on the surface of FL cells. Thus, expression of both CD70 and B7 co-stimulatory molecules appears to be essential for an efficient T-cell-mediated immune response. However, the molecules responsible for the significantly higher alloantigen T-cell response to FL cells activated through CD27 and CD40 remain to be identified. In summary, we conclude that CD27+CD70 represents another co-stimulatory pathway involved in T-cell-mediated immune responses to FL cells. Our findings suggest that several co-stimulatory pathways exist and should be taken into consideration to optimize antigen presentation for the generation of lymphoma-directed cytotoxic T cells for adoptive immunotherapy.
Assuntos
Antígenos CD , Linfoma Folicular/imunologia , Proteínas de Membrana/metabolismo , Linfócitos T/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo , Ligante CD27 , Antígenos CD40/metabolismo , Moléculas de Adesão Celular/metabolismo , Humanos , Ativação LinfocitáriaRESUMO
Persistent polyclonal B-cell lymphocytosis (PPBL) is a rare disorder of unknown aetiology affecting predominantly young to middle-aged women. It is characterized by a polyclonal expansion of B cells, including typical binucleated lymphocytes, and is associated with the presence of the translocation t(14;18), involving the bcl-2 oncogene. The stage of differentiation of the B cells expanded in PPBL is not known. We analysed the immunophenotype of the expanded B-cell subset in five new patients with PPBL and found a large uniform expansion of a recently defined human memory B-cell population, IgD(+)CD27(+) memory B cells. After in vitro stimulation with interleukin 2 (IL-2) and Staphylococcus aureus Cowan strain I, B cells from PPBL patients produced high levels of IgM immunoglobulins, which is a characteristic feature of IgD(+)CD27(+) memory B cells. Using a quantitative real-time polymerase chain reaction method, we found a high frequency of the translocation t(14;18) in the range of 1000-3000 per 106 B cells in PPBL patients. In contrast, a much smaller number of cells with a t(14;18) was found in B cells from healthy individuals. Our finding that PPBL is an accumulation of memory B cells further suggests that chronic antigeneic stimulation plays an important part in the pathogenesis of this disorder. This IgD(+)CD27(+) memory B-cell population might harbour a certain number of 'physiological' t(14;18) translocations that increases as this population expands in PPBL patients and constitutes the majority of peripheral blood lymphocytes.