Effects of Lipids on ENaC Activity in Cultured Mouse Cortical Collecting Duct Cells

Direct effects on epithelial Na⁺ channels (ENaC) activity by lipids, e.g., arachidonic acid (AA), eicosatetraynoic acid (ETYA), linoleic acid (LA), stearic acid (SA), hydroxyeicosatetraenoic acid (HETE), 11,12–epoxyeicosatrienoic acid (EET), (PGF2), and (PGE2), in cultured mouse cortical collecting duct (M1) cells were clarified by using single-channel recordings in this study. In a cell-attached recording, a bath application of 10 μM AA significantly reduced the ENaC open probability (NPo), whereas 10 μM ETYA or 5 μM LA only induced a slight inhibition. The inside-out recording as a standard protocol was thereafter performed to examine effects of these lipids on ENaC activity. Within 10 min after the formation of the inside-out configuration, the NPo of ENaC in cultured mouse cortical collecting duct (M1) cells remained relatively constant. Application of ETYA or LA or SA exhibited a similar inhibition on the channel NPo when applied to the extracellular side, suggesting that fatty acids could exert a nonspecific inhibition on ENaC activity. 11,12-EET, a metabolite of AA via the cytochrome P450 epoxygenase pathway, significantly inhibited the ENaC NPo, whereas 20-HETE, a metabolite of AA via the hydroxylase pathway, only caused a small inhibition of the ENaC NPo, to a similar degree as that seen with ETYA and LA. However, both PGE2 and PGF2α significantly enhanced the ENaC NPo. These results suggest that fatty acids exert a nonspecific effect on ENaC activity due to the interaction between the channel proximity and the lipid. The opposite effects of 11,12-EET and prostaglandin (PG) implicate different mechanisms in regulation of ENaC activity by activation of epoxygenase and cyclooxygenase.     

Collagen Synthesis in Mouse Embryonal Carcinoma Cells: Effect of Retinoic Acid

Abstract. In five lines of mouse embryonal carcinoma cells, PCC3/A1, PCC4, PCC4/Aza-R1, PCC7-S/Aza-R1, and F9, collagen synthesis was examined by immunofluorescence reaction using specific antibodies directed against collagen. All the embryonal carcinoma cell lines showed type IV collagen, and PCC7-S/Aza-R1 revealed the additional presence of type III collagen. When the F9 and PCC3/A1 EC cells were treated with retinoic acid and dibutyryl-cAMP, they differentiated into morphologically different cellular types. These cellular types showed new types of collagen. Thus, in treated F9 cells, type I, type III, and type V collagen were detected and in treated PCC3/A1 cells, type III and type V collagen were detected.
In two established cellular strains, PYS-2 corresponding to parietal endoderm and 3TDM-1 corresponding to trophoblastoma, collagen was identified by immunological reaction and electrophoretic mobility. The trophoblastoma cell line was characterized by the production of type I, type III, and type IV collagen, whereas endodermal PYS-2 revealed type IV collagen.     

Differential Effects of Aldosterone and Vasopressin on Chloride Fluxes in Transimmortalized Mouse Cortical Collecting Duct Cells

The effects of aldosterone and vasopressin on Cl⁻ transport were investigated in a mouse cortical collecting duct (mpkCCD) cell line derived from a transgenic mouse carrying the SV40 large T antigen driven by the proximal regulatory sequences of the L-pyruvate kinase gene. The cells had features of a tight epithelium and expressed the amiloride-sensitive sodium channel and the cystic fibrosis transmembrane conductance regulator (CFTR) genes. dD-arginine vasopressin (dDAVP) caused a rapid, dose-dependent, increase in short-circuit current (I _sc ). Experiments with ion channel blockers and apical ion substitution showed that the current represented amiloride-sensitive Na⁺ and 5-nitro-2-(3-phenylpropylamino)benzoate-sensitive and glibenclamide-sensitive Cl⁻ fluxes. Aldosterone (5 × 10⁻⁷ m for 3 or 24 hr) stimulated I _sc and apical-to-basal ²²Na⁺ flux by 3-fold. ³⁶Cl⁻ flux studies showed that dDAVP and aldosterone stimulated net Cl⁻ reabsorption and that dDAVP potentiated the action of aldosterone on Cl⁻ transport. Whereas aldosterone affected only the apical-to-basal ³⁶Cl⁻ flux, dDAVP mainly increased the apical-to-basal Cl⁻ flux and the basal-to-apical flux of Cl⁻ to a lesser extent. These results suggest that the discrete dDAVP-elicited Cl⁻ secretion involves the CFTR and that dDAVP and aldosterone may affect in different ways the observed increased Cl⁻ reabsorption in this model of mouse cultured cortical collecting duct cells. Received: 8 January 1998/Revised: 25 March 1998     

维甲酸促进小鼠F9细胞中STAT1的反式激活作用

维甲酸能促进肿瘤细胞的凋亡,诱导体外胚胎干细胞的分化,但作用机制的不明使其应用受到极大限制.因此,更多更全面地了解维甲酸的作用机制具有重要意义.本文构建了信号传导与转录激活因子（STAT1和STAT3）的真核表达载体 ,通过免疫荧光染色、电泳迁移率实验以及双荧光素酶报告基因检测系统证明, 维甲酸诱导可以激活转录因子STAT1促使其进入细胞核,并且增强STAT1蛋白与靶基因启动子的结合能力,从而发挥基因表达调控作用.本文结合后续的STAT1功能分析,试图建立起一种“维甲酸-转录因子-靶基因”的研究模式,有助于维甲酸作用机制的全面、系统的研究.这为临床上使用维甲酸作为抗肿瘤药提供理论基础,同时也为胚胎干细胞多能性调控机制研究提供新思路.     

Mechanism of Shrinkage Activation of Nonselective Cation Channels in M-1 Mouse Cortical Collecting Duct Cells

It has previously been shown that osmotic cell shrinkage activates a nonselective cation (NSC) channel in M-1 mouse cortical collecting duct cells [54] and in a variety of other cell types [20]. In the present study we further characterized the shrinkage-activated NSC channel in M-1 cells and its mechanism of activation using whole-cell current recordings. Osmotic cell shrinkage induced by addition of 100 mm sucrose to the bath solution caused a 20-fold increase in whole-cell inward currents from −10.8 ± 1.5 pA to −211 ± 10.2 pA (n= 103). A similar response was observed when cell shrinkage was elicited using a hypo-osmotic pipette solution. This indicates that cell shrinkage and not extracellular osmolarity per se is the signal for current activation. Cation substitution experiments revealed that the activated channels discriminate poorly between monovalent cations with a selectivity sequence NH₄ (1.2) ≥ Na⁺ (1) ≈ K⁺ (0.9) ≈ Li⁺ (0.9). In contrast there was no measurable permeability for Ca²⁺ or Ba²⁺ and the cation-to-anion permeability ratio was about 14. The DPC-derivatives flufenamic acid, 4-methyl-DPC and DCDPC were the most effective blockers followed by LOE 908, while amiloride and bumetanide were ineffective. The putative channel activator maitotoxin had no effect. Current activation was dependent upon the presence of intracellular ATP and Mg²⁺ and was inhibited by staurosporine (1 μm) and calphostin C (1 μm). Moreover, cytochalasin D (10 μm) and taxol (2 μm) reduced the current response to cell shrinkage. These findings suggest that the activation mechanism of the shrinkage-activated NSC channel involves protein kinase mediated phosphorylation steps and cytoskeletal elements. Received: 3 May 2000/Revised: 6 July 2000     

Thymus-derived Cells in Mouse Thoracic Duct Lymph

Thymus lymphocytes injected into neo-natally thymectomized mice were identified in the thoracic duct lymph by their θ antigens and shown to form part of the recirculating lymphocyte pool.     

PLK1 Signaling in Breast Cancer Cells Cooperates with Estrogen Receptor-Dependent Gene Transcription

1. Download : Download full-size image

     

Retinoic Acid Treatment Induces Cell Death and the Protein Expression of Retinoic Acid Receptor β in the Mesenchymal Cells of Mouse Facial Primordia in Vitro

We isolated mesenchymal cells from individual facial primordia of mouse embryos on 11 days post coitum and examined the effects of retinoic acid (RA) on chondrogenesis, induction of cell death, and the protein expression of retinoic acid receptor (RAR) β and γ in micromass culture. Under the control condition, cells of both medial and lateral nasal prominences (MNP and LNP) displayed high chondrogenic potential, while those of maxillary and mandibular prominences (Mx and Md) had constant growth activity and low chondrogenic potential. Though none of the cells expressed detectable levels of the RAR β protein, RAR γ was expressed in the cells of all the facial primordia. One μM RA inhibited the chondrogenesis, and induced cell death accompanied with the induction of the RAR β protein in LNP, MX and Md cells within 6 hr. On the contrary, both cell death and RAR β protein induction were detected in the MNP cells treated with RA for 24 hr. These results suggest that the RAR β is involved in the process of the cell death induced by the RA treatment in the mesenchymal cells of the mouse facial primordia.     

Insulin Regulates Nitric Oxide Production in the Kidney Collecting Duct Cells

The kidney is an important organ for arterial blood pressure (BP) maintenance. Reduced NO generation in the kidney is associated with hypertension in insulin resistance. NO is a critical regulator of vascular tone; however, whether insulin regulates NO production in the renal inner medullary collecting duct (IMCD), the segment with the greatest enzymatic activity for NO production in kidney, is not clear. Using an NO-sensitive 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) fluorescent dye, we found that insulin increased NO production in mouse IMCD cells (mIMCD) in a time- and dose-dependent manner. A concomitant dose-dependent increase in the NO metabolite (NOx) was also observed in the medium from insulin-stimulated cells. NO production peaked in mIMCD cells at a dose of 100 nm insulin with simultaneously increased NOx levels in the medium. At this dose, insulin significantly increased p-eNOS^Ser1177 levels in mIMCD cells. Pretreatment of cells with a PI 3-kinase inhibitor or insulin receptor silencing with RNA interference abolished these effects of insulin, whereas insulin-like growth factor-1 receptor (IGF-1R) silencing had no effect. We also showed that chronic insulin infusion to normal C57BL/6J mice resulted in increased endothelial NOS (eNOS) protein levels and NO production in the inner medulla. However, insulin-infused IRKO mice, with targeted deletion of insulin receptor from tubule epithelial cells of the kidney, had ∼50% reduced eNOS protein levels in their inner medulla along with a significant rise in BP relative to WT littermates. We have previously reported increased baseline BP and reduced urine NOx in IRKO mice. Thus, reduced insulin receptor signaling in IMCD could contribute to hypertension in the insulin-resistant state.     

前列腺特异启动子和部分靶基因在转基因模型中的应用进展

遗传工程小鼠模型已被大量用于前列腺癌发生、发展、恶化和转移的研究。构建小鼠模型的途径主要包括转基因的过表达、基因敲除或敲入,以及使用Cre/lox技术进行条件调控。本文综述了目前构建的有明显遗传表型的转基因小鼠模型所用到的前列腺特异的启动子,以及所用到的靶基因。