Cystic fibrosis transmembrane conductance regulator and the outwardly rectifying chloride channel: a relationship between two chloride channels expressed in epithelial cells

1. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) result in the primary defect observed in patients with cystic fibrosis. 2. The CFTR is a member of the ATPase-binding cassette (ABC) transporter family but, unlike other members of this group, CFTR conducts a chloride current that is activated by cAMP. 3. In epithelial cells, the cAMP-stimulated chloride current is conducted by both CFTR and the outwardly rectifying chloride channel (ORCC). 4. The present review summarizes the current knowledge of the properties of the two channels, as well as their relationship. Because the gene encoding the ORCC has not been identified, a discussion as to possible candidates for this chloride channel is included.     

拓扑异构酶Ⅱ抑制剂诱导SUNE-1鼻咽癌细胞凋亡的研究

目的:探讨拓扑异构酶Ⅱ(TopoⅡ)抑制剂吡喃阿霉素(THP)、阿霉素(ADM)诱导鼻咽低分化鳞癌细胞株SUNE-1细胞凋亡的作用;并检测药物作用后SUNE-1线粒体膜电位及Livin表达水平的变化。方法:采用MTT法检测THP、ADM对SUNE-1细胞的生长抑制,Annenxin V FITC染色检测凋亡早期细胞,流式细胞仪检测线粒体膜电位改变和Livin的表达情况。结果:ADM和THP对SUNE-1具有较强的体外抗肿瘤作用,随着药物浓度增高细胞增殖抑制率增高,其IC50分别为0.004 1±0.000 3"g/ml和0.003 8±0.000 8"g/ml(P=0.542)。随着药物作用时间的增加,SUNE-1细胞凋亡率逐渐增高,同时伴有线粒体内膜电负性逐渐增加。ADM、THP诱导SUNE-1细胞凋亡中出现Livin的表达上调,但THP作用后表达上调的程度较ADM低。结论:TopoⅡ抑制剂蒽环类药物THP、ADM具有抑制细胞增殖和诱导鼻咽低分化鳞癌细胞株SUNE-1细胞凋亡的作用。TopoⅡ抑制剂作用鼻咽癌细胞后诱导IAP蛋白中的Livin表达上调,但THP作用后所引起的Livin表达上调不如ADM显著,因此THP在治疗鼻咽癌时可能具有更好的应用价值,需在临床中进一步证实。     

Calcium ionophoretic and apoptotic effects of ferutinin in the human Jurkat T-cell line

We have investigated the ionophoretic and apoptotic properties of the daucane sesquiterpene ferutinin and three related compounds, ferutidin, 2-alpha-hydroxyferutidin and teferin, all isolated from various species of plants from the genus Ferula. Ferutinin induced a biphasic elevation of intracellular Ca2+ in the leukemia T-cell line, Jurkat. First, a rapid calcium peak was observed and inhibited by BAPTA-AM. This initial calcium mobilization was followed by a sustained elevation, mediated by the entry of extracellular calcium through L-type calcium channels and sensitive to inhibition by EGTA. Moreover, ferutinin-induced apoptosis in Jurkat cells, and this event was preceded, in a cyclosporine-A sensitive manner, by a loss of mitochondrial transmembrane potential (DeltaPsim) and by an increase in intracellular reactive oxygen species. Ferutinin-induced DNA fragmentation was mediated by a caspase-3-dependent pathway, and was initiated independently of any specific phase of the cell cycle. The evaluation of ferutinin analogs in calcium mobilization and apoptosis assays showed strict structure-activity relationships, with p-hydroxylation of the benzoyl moiety being requested for activity.     

Reversal of antigen-dependent signaling by two mutations in antibody/receptor chimera: implication of inverse agonism in cytokine receptor superfamily

Understanding the receptor activation mechanism is essential for the rational design of pharmacologically active ligand molecules. However, the activation mechanism of most cytokine receptors remains still unclear, and while agonism and antagonism have been described for ligand-mimetic peptides, there has been no report of inverse agonism that has been characterized for G protein-coupled receptors (GPCRs). To explore the activation mechanism of cytokine receptors, here we tried to investigate how agonism and antagonism could be altered by randomizing antibody variable region of an antibody/cytokine receptor chimera recognizing hen egg lysozyme (HEL) as an agonist. Based on our previous finding that the co-expression of V(H)-gp130 and V(L)-erythropoietin receptor (EpoR) chimeras transduced strict and efficient HEL-dependent cell growth signal, a V(H)-gp130 library encoding four randomized CDR2 residues was retrovirally infected to IL-3-dependent Ba/F3 cells already transfected with V(L)-EpoR. The selection without IL-3 resulted in a clonal expansion of the transduced cells, and interestingly some of which showed HEL dose-dependent growth suppression. Our results clearly indicate that agonism and antagonism of the antibody/cytokine receptor chimera can be readily switched by a subtle modification of the ligand binding domain as well as that of GPCRs, also implying the existence of inverse agonism in cytokine receptor superfamily.     

Differences in binding properties of two proton pump inhibitors on the gastric H+,K+-ATPase in vivo

Restoration of acid secretion after treatment with covalently-bound proton pump inhibitors may depend on protein turnover and on reversal of inhibition by reducing agents such as glutathione. Glutathione incubation of the H(+),K(+)-ATPase isolated from omeprazole or pantoprazole-treated rats reversed 88% of the omeprazole inhibition but none of the pantoprazole inhibition. The present study was designed to measure binding properties of omeprazole or pantoprazole in vivo. Rats were injected with (14)C-omeprazole or (14)C-pantoprazole after acid stimulation. The specific binding to the gastric H(+),K(+)-ATPase was measured at timed intervals as well as reversal of binding by glutathione reduction. The stoichiometry of omeprazole and pantoprazole binding to the catalytic subunit of the H(+),K(+)-ATPase was 2 moles of inhibitor per mole of the H(+),K(+)-ATPase phosphoenzyme. Omeprazole bound to one cysteine between transmembrane segments 5/6 and one between 7/8, pantoprazole only to the two cysteines in the TM5/6 domain. Loss of drug from the pump was biphasic, the fast component accounted for 84% of omeprazole binding and 51% of pantoprazole binding. Similarly, only 16% of omeprazole binding but 40% of pantoprazole binding was not reversed by glutathione. The residence time of omeprazole and pantoprazole on the ATPase in vivo depends on the reversibility of binding. Binding of pantoprazole at cysteine 822 is irreversible whereas that of omeprazole at cysteine 813 and 892 is reversible both in vivo and in vitro. This is consistent with the luminal exposure of cysteine 813 and 892 and the intra-membranal location of cysteine 822 in the 3D structure of the H(+),K(+)-ATPase.     

Mutagenesis studies of the human MT2 melatonin receptor

Melatonin mediates its physiological effects through activation of high affinity G protein-coupled receptors. The vertebrate MT(1), MT(2) and Mel(1c) melatonin receptors are molecularly and pharmacologically distinct. Three molecular models of melatonin recognition for the MT(1) and/or Mel(1c) melatonin receptors have been proposed. To determine if these models applied to the MT(2) melatonin receptor, we mutated seven conserved residues to alanine in the hMT(2) melatonin receptor and expressed the receptors in HEK-293 cells. Competition of melatonin for 2-[125I]-iodomelatonin binding revealed that mutation of Asn 16 in TM4 or His 7 in TM5 of the hMT(2) melatonin receptor significantly decreased the binding affinity for melatonin when compared with wild-type. In addition, competition of 4P-ADOT, N-acetyltryptamine, luzindole, and 5-methoxytryptophol for 2-[125I]-iodomelatonin binding suggested Asn 16 in TM4 may facilitate binding of the 5-methoxy group of the melatonin molecule to the hMT(2) melatonin receptor. Trp 13 or Phe 6 in TM6 while not critical for melatonin binding, may interact with aromatic regions of luzindole and 4P-ADOT. Mutation of Ser 8 or Ser 12 in TM3, or Ser 6 in TM7 did not affect the affinity of melatonin for competition with 2-[125I]-iodomelatonin to the hMT(2) melatonin receptor, although equivalent serines (Ser 8 and Ser 12 in TM3) were reported to be critical for melatonin binding to the hMT(1) melatonin receptor. Thus these results are the first to identify residues within the transmembrane regions of the hMT(2) melatonin receptor critical for melatonin binding, highlighting potential structural differences between the MT(1) and MT(2) melatonin receptor binding pockets.     

A single nucleotide polymorphism in the transmembrane domain coding region of HER-2 is associated with development and malignant phenotype of gastric cancer

Alterations of the HER-2 (erbB-2/neu) proto-oncogene have been associated with carcinogenesis and poor prognosis of certain cancers. A single nucleotide polymorphism (Ile/Val, A/G) in the transmembrane domain was reported to be associated with a risk of breast cancer. In our study, we examined the association between the HER-2 polymorphism and gastric carcinoma. The Ile/Ile, Ile/Val and Val/Val genotypes were found in 146 (68.9%), 56 (26.4%) and 10 (4.7%) of 212 gastric cancer patients and in 234 (81.5%), 48 (16.7%) and 5 (1.8%) of 287 control subjects, respectively. The Ile/Val or Val/Val genotype was significantly more frequent in patients than in controls (p = 0.005 and 0.033, respectively). The OR of Val/Val genotype then revealed a significantly enhanced risk of 3.25 (95% CI 1.09-9.70) compared to Ile/Ile genotype; heterozygous Ile/Val genotype showed an intermediate risk of 1.97 (1.27-3.06). In patients, carcinomas of advanced stage were significantly more frequent in patients with Ile/Val or Val/Val genotype than those with Ile/Ile genotype (p < 0.001). The logistic regression analysis for tumor invasion, lymph node metastasis and distant metastasis revealed that lymph node metastasis was most closely associated with the HER-2 genotype. These results suggest that this nucleotide polymorphism in the transmembrane domain-coding region of HER-2 could be associated with development of gastric carcinoma and may serve as a predictor of risk for a malignant phenotype of gastric cancer. The association of HER-2 genotype with clinicopathologic characteristics of gastric cancer was also suggested, which has to be confirmed with a larger sample size.     

Regulation of proliferation, survival and apoptosis by members of the TNF superfamily

Tumor necrosis factor (TNF) was first identified in 1984 as a cytokine with anti-tumor effects in vitro and in vivo. Extensive research since then has shown that there are at least 18 distinct members of the TNF super family and they exhibit 15-25% amino acid sequence homology with each other. These family members bind to distinct receptors, which are homologous in their extracellular domain. These cytokines have been implicated in a wide variety of diseases including tumorigenesis, septic shock, viral replication, bone resorption, rheumatoid arthritis, diabetes, and other inflammatory diseases. TNF blockers have been approved for human use in treating some of these conditions in the United States and other countries. Various members of the TNF super family mediate either proliferation, survival, or apoptosis of cells. Although distinct receptors, all members share a common cell signaling pathway that mediates the activation of nuclear factor-kappaB (NF-kappaB) and mitogen-activated protein kinases (e.g. c-jun N-terminal kinase). Regulation of cell growth and activation of NF-kappaB and of c-jun N-terminal kinase by the TNF super family is mediated through sequential activation/association of a set of cell signaling proteins named TNF receptor-associated factors, Fas-associated death domain and FADD-like ICE, caspases, receptor-interacting protein, NF-kappaB-inducing kinases, and IkappaBalpha kinases. Both apoptotic and antiapoptotic signals are activated simultaneously by the same cytokine in the same cell. Together these cytokines regulate cell growth/survival/apoptosis in a complex dance of changing partners and overlapping steps.     

Mitochondrion-dependent caspase activation by the HIV-1 envelope

Cells expressing the envelope glycoprotein complex (Env) encoded by the human immunodeficiency virus can fuse with cells expressing Env receptors (CD4 and CXCR4). The resulting syncytia undergo apoptosis. We developed a cytofluorometric assay for the quantitation of syncytium formation and syncytial apoptosis. Using this methodology, we show that caspase activation in syncytia is inhibited by pharmacological or genetic intervention on cyclin-dependent kinase-1, p53, and mitochondrial membrane permeabilization (MMP). Thus, transfection of fusing cells with the viral mitochondrial inhibitor of apoptosis encoded by cytomegalovirus, a specific inhibitor of MMP, prevented the mitochondrial cytochrome c release and abolished simultaneously the activation of caspase-3. Conversely, inhibition of caspases did not prevent MMP. These results indicate that Env-elicited syncytial apoptosis involves the intrinsic (mitochondrial) pathway.     

Peptide and nonpeptide antagonist interaction with constitutively active human AT1 receptors

Wild type human AT(1) receptors (WT-AT(1)) and mutant receptors, in which Asn(111) was replaced by glycine (N111G), alanine (N111A) and serine (N111S), or in which Asp(281) was replaced by alanine (D281A) or in which N111G and D281A replacements were combined, were transiently expressed in CHO-K1 cells. While the biphenyltetrazole compound candesartan dissociated slowly and behaved as an insurmountable antagonist for WT-AT(1), it dissociated swiftly and only produced a rightward shift of the angiotensin Ang II- and -IV dose-response curves for inositol phosphate (IP) accumulation in cells expressing N111G. [3H]candesartan competition binding yielded the same potency order of the related biphenyltetrazoles for WT-AT(1) and mutated receptors, i.e. candesartan>EXP3174>irbesartan>losartan. Affinities were equal for WT-AT(1) and D281A and 40- to 400-fold lower for all Asn(111) mutants. Mutations did not affect the affinity of the peptide antagonist [Sar(1)Ile(8)]Ang II (SARILE). Basal IP accumulation in cells with WT-AT(1) was not affected by any biphenyltetrazole antagonists and was increased by SARILE to 19% of the maximal Ang II stimulation. Basal IP accumulation was higher for cells expressing the Asn(111)-mutated receptors. For N111G, this accumulation was partially inhibited by all the biphenyltetrazoles upon long-term (18hr) exposure. In these cells SARILE produced the same maximal stimulation as Ang II. Asn(111)-mutated AT(1) receptors are thought to mimic the pre-activated state of the wild type receptor and comparing the efficacy and affinity of ligands for such mutated receptors facilitate the distinction of partial (SARILE) and inverse (biphenyltetrazoles) agonists from true antagonists.