The Functional and Molecular Entities Underlying Amino Acid and Peptide Transport By the Mammary Gland Under Different Physiological And Pathological Conditions

This review describes the properties and regulation of the membrane transport proteins which supply the mammary gland with aminonitrogen to support metabolism under different physiological conditions (i.e. pregnancy, lactation and involution). Early studies focussed on characterising amino acid and peptide transport pathways with respect to substrate specificity, kinetics and hormonal regulation to allow a broad picture of the systems within the gland to be established. Recent investigations have concentrated on identifying the individual transporters at the molecular level (i.e. mRNA and protein). Many of the latter studies have identified the molecular correlates of the transport systems uncovered in the earlier functional investigations but in turn have also highlighted the need for more amino acid transport studies to be performed. The transporters function as either cotransporters and exchangers (or both) and act in a coordinated and regulated fashion to support the metabolic needs of the gland. However, it is apparent that a physiological role for a number of the transport proteins has yet to be elucidated. This article highlights the many gaps in our knowledge regarding the precise cellular location of a number of amino acid transporters within the gland. We also describe the role of amino acid transport in mammary cell volume regulation. Finally, the important role that individual mammary transport proteins may have in the growth and proliferation of mammary tumours is discussed.     

Emerging role of AMP-activated protein kinase in coupling membrane transport to cellular metabolism

PURPOSE OF REVIEW: It has long been recognized that the coupling of membrane transport to underlying cellular metabolic status is critical because transport processes consume a large portion of total cellular energy. Recently, the finely tuned metabolic sensor AMP-activated protein kinase (AMPK) has emerged as a membrane transport regulator, which may permit sensitive transport-metabolism crosstalk. This review will discuss how AMPK may play an important role in the regulation of ion and solute transport across the plasma membrane under both physiological and pathological conditions in epithelia and other tissues. RECENT FINDINGS: Recent studies have found that AMPK, which becomes activated during cellular metabolic stress, promotes the cellular uptake of fuel sources such as glucose and fatty acids to promote ATP generation and inhibits ion-transport proteins such as the cystic fibrosis transmembrane conductance regulator Cl channel and the epithelial Na channel, thereby limiting the dissipation of transmembrane ion gradients. An understanding of the underlying cellular and molecular mechanisms for AMPK-dependent regulation of transport proteins is beginning to emerge. SUMMARY: As earlier studies have focused on the role of nucleotides such as ATP in regulating transport-protein activities, the regulation of membrane transport by AMPK represents a novel and more-sensitive mechanism for the coupling of membrane transport to cellular metabolic status. Identifying new membrane-transport targets of AMPK and elucidating the mechanisms involved in their AMPK-dependent regulation are fruitful areas for new investigation that should yield valuable insights into the pathophysiology of hypoxic and ischemic tissue injury.     

PDZ proteins and proximal ion transport

PURPOSE OF REVIEW: PDZ proteins are major structural components of protein assembly. This review covers the implications of these proteins in the regulation of transport systems expressed in renal proximal tubules. RECENT FINDINGS: In the last few years, many reports have highlighted the implication of PDZ proteins in two aspects of proximal tubule physiology, namely the generation and maintenance of epithelial polarity and the formation of regulatory complexes that provide spatial and molecular specificity to the intracellular signalling. SUMMARY: PDZ-mediated interactions are involved in a wide range of cellular functions, from cell division to cell polarity to intracellular signalling. Consistent with this functional spectrum, ablation of PDZ protein genes generates a wide panel of pathological phenotypes, some of which link directly to human syndromes. In proximal tubules, PDZ proteins are thought to play a major role in epithelial polarity and transport regulation.     

The regulation of renal phosphate transport

Renal phosphate transport is mediated by the abundance and activity of the sodium-dependent phosphate transporters, Npt2a, Npt2c, and PiT-2, present within the apical brush border membrane of the proximal tubule. Recent studies have demonstrated differential expression and activity of these sodium-dependent phosphate transporters within the proximal tubule. In general, phosphate transport is regulated by a variety of physiological stimuli, including parathyroid hormone, glucocorticoids, vitamin D3, estrogen, and thyroid hormone. Phosphatonins are now recognized as major regulators of phosphate transport activity. Other factors that affect phosphate transport include dopamine, dietary phosphate, acid-base status, lipid composition, potassium deficiency, circadian rhythm, and hypertension. Studies have shown that the PDZ-containing sodium/hydrogen exchanger regulatory factor (NHERF) proteins, specifically NHERF-1 and NHERF-3, play a critical role in the physiological regulation of phosphate transport, particularly in response to dietary phosphate. In addition, recent studies have found that NHERF-1 is also important in both the parathyroid hormone- and dopamine-mediated inhibition of phosphate transport. This review will detail the various hormones and agents involved in the regulation of phosphate transport as well as provide a brief summary of the signaling pathways and cytoskeletal proteins active in the transport of phosphate in the renal proximal tubule.     

Interaction of syntaxins with epithelial ion channels

Recent evidence suggests that some of the syntaxin isoforms may physically interact with and regulate the transport activity of a defined set of membrane transport proteins. This review examines recent studies of the cystic fibrosis transmembrane conductance regulator and the epithelial sodium channel which define distinct roles of syntaxin 1A and syntaxin 3 in the regulation of surface expression as well as intrinsic properties of these epithelial ion transporters.     

Molecular approaches to urea transporters

Urea plays a critical role in the urine-concentrating mechanism in the inner medulla. Physiologic data provided evidence that urea transport in red blood cells and kidney inner medulla was mediated by specific urea transporter proteins. Molecular approaches during the past decade resulted in the cloning of two gene families for facilitated urea transporters, UT-A and UT-B, encoding several urea transporter cDNA isoforms in humans, rodents, and several nonmammalian species. Polyclonal antibodies have been generated to the cloned urea transporter proteins, and the use of these antibodies in integrative animal studies has resulted in several novel findings, including: (1) the surprising finding that UT-A1 protein abundance and urea transport are increased in the inner medulla during conditions in which urine concentrating ability is reduced; (2) vasopressin increases UT-A1 phosphorylation in rat inner medullary collecting duct; (3) UT-A protein abundance is upregulated in uremia in both liver and heart; and (4) UT-B is expressed in many nonrenal tissues and endothelial cells. This review will summarize the knowledge gained from using molecular approaches to perform integrative studies into urea transporter protein regulation, both in normal animals and in animal models of human diseases, including studies of uremic rats in which urea transporter protein is upregulated in liver and heart.     

Oxalate transport and calcium oxalate renal stone disease

Hyperoxaluria is considered to play a crucial role in calcium oxalate (CaOx) renal stone disease. The amount of oxalate excreted into the urine depends on intestinal absorption, endogenous production, renal clearance and renal tubular transport. Since a primary disorder has not been found so far in most CaOx stone formers and since oxalate is freely filtered at the glomerulus, most studies are presently focussed on alterations in epithelial oxalate transport pathways. Oxalate can be transported across an epithelium by the paracellular (passive) and transcellular (active) pathway. Oxalate transport across cellular membranes is mediated by anion-exchange transport proteins. A defect in the structure of these transport proteins could explain augmented transcellular oxalate transport. Little is known about the physiological regulation of oxalate transport. In this review cellular transport systems for oxalate will be summarized with special attention for the progress that has been made to study oxalate transport in a model of cultured renal tubule cells. Better understanding of the physiological processes that are involved in oxalate transport could yield information on the basis of which it might be possible to design new approaches for an effective treatment of CaOx stone disease.     

Molecular aspects in the regulation of renal inorganic phosphate reabsorption: the type IIa sodium/inorganic phosphate co-transporter as the key player

The type IIa sodium/inorganic phosphate co-transporter is the rate-limiting inorganic phosphate transport pathway in renal brush-border membranes, and is thus a key player in overall inorganic phosphate homeostasis. Its regulation is mostly associated with membrane retrieval/reinsertion (traffic) of the transport protein. This membrane traffic is controlled by specific 'motifs' at the level of the transporter protein and probably involves interacting proteins (e.g. for scaffolding, regulation or sorting). The intracellular signaling mechanisms (e.g. the involvement of kinases) and the involvement of the cytoskeleton are not yet understood. Hereditary alterations in renal inorganic phosphate handling can be associated with factors controlling the expression of the brush-border type IIa sodium/inorganic phosphate co-transporter.     

Renal phosphate-wasting disorders

The renal regulation of phosphate is a complex process. Clinical disorders of phosphate handling have led to the identification of several genes and proteins involved in the maintenance of phosphate homeostasis. Further work is required to elucidate the precise pathways that regulate renal phosphate transport.     

Lessons from mouse mutants of epithelial sodium channel and its regulatory proteins

The use of gene-modified mouse models allows the experimental in vivo analysis of specific gene defects at the level of target cells. With respect to the epithelial sodium channel and some of its regulatory proteins, gene-modified models that control gene defects in a time- and tissue-dependent conditional or constitutive manner have been generated. The combination of molecular and physiologic approaches in these mouse models increases the understanding of the complex regulation and the cell signaling cascades involved in Na(+) transport in target cells and may ultimately provide new insights into the pathophysiology of renal Na(+) retention and BP regulation. This review summarizes and discusses the gene-targeting approaches that have been applied to the epithelial sodium channel and its regulatory proteins.     

Sodium reabsorption in aldosterone-sensitive distal nephron: news and contributions from genetically engineered animals

The precise adaptation of renal sodium excretion to systemic needs is to a large extent achieved by the regulation of sodium re-absorption in the aldosterone-sensitive distal nephron. Transcellular sodium re-absorption by the segment-specific cells of the aldosterone-sensitive distal nephron (often called principal cells) is mainly controlled at the level of the expression and activity levels of the epithelial sodium channel, the apical amiloride-sensitive sodium influx pathway. Recent investigations have identified the first early aldosterone-induced proteins that act on epithelial sodium channel function in expression systems. Indirect evidence suggests that one of these aldosterone-induced proteins, the serum- and glucocorticoid-inducible protein kinase SGK1, plays a central integratory role in the control of epithelial sodium channel surface expression and activity, also in the mammalian kidney. Gene-modified animals lacking epithelial sodium channel subunits or expressing mutant subunits have substantiated the central role of the epithelial sodium channel in sodium re-absorption and blood pressure control, as well as for neonatal lung liquid clearance. Mice overexpressing or lacking specific hormones or their receptors have been used to study their role in sodium transport regulation, but the study of mouse physiology appears to lag behind the generation of gene-modified mice. Nonetheless, these new animal models have had a strong impact on research, by stimulating the integration of knowledge and techniques learned from reductionistic molecular approaches into tissue and animal studies, thus breaking down barriers and stimulating collaborations.     

高通量互作蛋白组图谱研究揭示U6 snRNA在肿瘤细胞中多维度调控mRNA加工成熟过程

目的 采用ChIRP技术纯化肿瘤细胞中内源U6 snRNA（简称U6）互作蛋白,结合高灵敏度液相色谱-质谱联用技术对所获得的互作蛋白组进行鉴定,系统揭示U6参与肿瘤细胞中新的生物学功能和分子调控机制。方法 合成含生物素标记的U6特异性探针,利用分子杂交技术特异捕获经甲醛交联的内源U6与互作蛋白形成的复合物,并结合高灵敏度质谱分析技术鉴定出U6互作蛋白组。利用生物信息分析手段分析U6在肿瘤细胞中的功能。结果 通过严格的筛选本研究最终鉴定到135个特异的内源U6互作蛋白。GO、KEGG、蛋白复合体富集分析和PPI分析显示U6除了与mRNA前体剪接加工等相关蛋白发生相互作用外,还参与了mRNA从产生到转录后调控等一系列生物学过程,包括参与mRNA转录调控、囊泡运输、成熟mRNA的出核转运、mRNA降解、RNA定位、转录后调控等过程。结论 我们的研究在国际上首次系统揭示了肿瘤细胞U6的相互作用蛋白组图谱,通过对该图谱的分析表明U6多维度地参与了mRNA生物学发生的全过程调控,为U6参与肿瘤发生发展的功能和机制研究提供了全新的方向和思路。     

Hernia disease and collagen gene regulation: are there clues for intervention?

Collagens belong to the most abundant proteins in the body. After tissue injury, a coordinated regulation of collagen gene expression guides the formation of a provisional matrix that subsequently evolves into a mature scar with tensile strength. In the following, knowledge regarding collagen gene regulation that may provide insight into how to specifically address the biological problem of soft tissue weakness and recurrent hernia disease is summarized.     

The roles and mechanisms of intestinal oxalate transport in oxalate homeostasis

The mammalian intestine has an important role in the dynamics of oxalate exchange and thereby is significant in the etiology of calcium oxalate nephrolithiasis. Here we review some of the phenomenologic observations that have led to the conclusion that anion exchangers (antiporters) are important mediators of secondarily active, net oxalate transport along the intestine (both absorptive and secretory). Understanding the mechanisms of transepithelial oxalate transport has been advanced radically in recent years by the identification of the solute-linked carrier (SLC)26 family of anion transporters, which has facilitated the identification of specific proteins mediating individual apical or basolateral oxalate transport pathways. Moreover, identification of specific exchangers has underscored their relative importance to oxalate homeostasis as revealed by using knockout mouse models and has facilitated studies of oxalate transport regulation in heterologous expression systems. Finally, the significance of oxalate degrading bacteria to oxalate homeostasis is considered from basic and applied perspectives.     

Changing subcellular localization of nuclear transport factors during human spermatogenesis

Spermatogenesis requires progressive changes in gene expression mediated by hormonal and local factors. Regulated macromolecular movement between nuclear and cytoplasmic compartments enables these essential responses to changing extracellular cues, and dynamic production of the nucleocytoplasmic transporters and importin proteins, throughout gametogenesis in rodents implicates them as key mediators of germline differentiation. We examined normal adult human testis expression profiles of six importins plus five additional proteins involved in nucleocytoplasmic transport. Although most were detected in the nucleus during germline differentiation, importin α4 was exclusively observed in Sertoli and germ cell cytoplasm. Many proteins were present in round spermatid nuclei (importins α1, α3, β1, β3; exportin-1, Nup62, Ran, RanBP1, RCC1), and remarkable intense nuclear and/or nuclear-associated signals were detected for importin α1, importin α3 and Nup62 in spermatocytes. This study identifies conserved aspects of nucleocytoplasmic transport during spermatogenesis and extends our knowledge of the dynamic presence of these proteins, which indicates that they contribute to germ cell-specific cargo trafficking and potentially to other functions during human spermatogenesis. We also demonstrate for the first time that importin α3 is nuclear in spermatocytes, when exportin-1 is cytoplasmic, suggesting that nuclear transport is altered during meiosis.     

Uncoupling proteins 2 and 3: potential regulators of mitochondrial energy metabolism

Mitochondria use energy derived from fuel combustion to create a proton electrochemical gradient across the mitochondrial inner membrane. This intermediate form of energy is then used by ATP synthase to synthesize ATP. Uncoupling protein-1 (UCP1) is a brown fat-specific mitochondrial inner membrane protein with proton transport activity. UCP1 catalyzes a highly regulated proton leak, converting energy stored within the mitochondrial proton electrochemical potential gradient to heat. This uncouples fuel oxidation from conversion of ADP to ATP. In rodents, UCP1 activity and brown fat contribute importantly to whole-body energy expenditure. Recently, two additional mitochondrial carriers with high similarity to UCP1 were molecularly cloned. In contrast to UCP1, UCP2 is expressed widely, and UCP3 is expressed preferentially in skeletal muscle. Biochemical studies indicate that UCP2 and UCP3, like UCP1, have uncoupling activity. While UCP1 is known to play an important role in regulating heat production during cold exposure, the biological functions of UCP2 and UCP3 are unknown. Possible functions include 1) control of adaptive thermogenesis in response to cold exposure and diet, 2) control of reactive oxygen species production by mitochondria, 3) regulation of ATP synthesis, and 4) regulation of fatty acid oxidation. This article will survey present knowledge regarding UCP1, UCP2, and UCP3, and review proposed functions for the two new uncoupling proteins.     

Molecular pathophysiology of tubular transport disorders

Inherited tubular transport disorders comprise a group of diseases that lead to profound derangements in the homeostasis of electrolytes, minerals, or organic solutes in the body. In the past decade remarkable progress has been made in our understanding of the molecular pathogenesis of hereditary tubulopathies and the fundamental molecular physiology of renal tubular transport processes. This review summarizes hereditary diseases caused by mutations in genes encoding transporter or channel proteins operating along the renal tubule. Review of the molecular basis of hereditary tubulopathies reveals various loss-of-function or gain-of-function mutations in genes encoding cotransporter, exchanger, or channel proteins, which are located in the luminal, basolateral, or endosomal membranes of the tubular cell or in paracellular tight junctions. These gene mutations result in a variety of functional defects in transporter/channel proteins, including decreased activity, impaired gating, defective trafficking, impaired endocytosis and degradation, or defective assembly of channel subunits. Further molecular studies of inherited tubular transport disorders may shed more light on the molecular pathophysiology of these diseases and may significantly improve our understanding of the mechanisms underlying renal salt homeostasis, urinary mineral excretion, and blood pressure regulation in health and disease. The identification of the molecular defects in inherited tubulopathies may provide a basis for future design of targeted therapeutic interventions and, possibly, strategies for gene therapy of these complex disorders. Received: 8 January 2001 / Revised: 6 June 2001 / Accepted: 6 June 2001     

Acid-base status determines the renal expression of Ca2+ and Mg2+ transport proteins

Chronic metabolic acidosis results in renal Ca2+ and Mg2+ wasting, whereas chronic metabolic alkalosis is known to exert the reverse effects. It was hypothesized that these adaptations are mediated at least in part by the renal Ca2+ and Mg2+ transport proteins. The aim of this study, therefore, was to determine the effect of systemic acid-base status on renal expression of the epithelial Ca2+ channel TRPV5, the Ca2+-binding protein calbindin-D28K, and the epithelial Mg2+ channel TRPM6 in relation to Ca2+ and Mg2+ excretion. Chronic metabolic acidosis that was induced by NH4Cl loading or administration of the carbonic anhydrase inhibitor acetazolamide for 6 d enhanced calciuresis accompanied by decreased renal TRPV5 and calbindin-D28K mRNA and protein abundance in wild-type mice. In contrast, metabolic acidosis did not affect Ca2+ excretion in TRPV5 knockout (TRPV5-/-) mice, in which active Ca2+ reabsorption is effectively abolished. This demonstrates that downregulation of renal Ca2+ transport proteins is responsible for the hypercalciuria. Conversely, chronic metabolic alkalosis that was induced by NaHCO3 administration for 6 d increased the expression of Ca2+ transport proteins accompanied by diminished urine Ca2+ excretion in wild-type mice. However, this Ca2+-sparing action persisted in TRPV5-/- mice, suggesting that additional mechanisms apart from upregulation of active Ca2+ transport contribute to the hypocalciuria. Furthermore, chronic metabolic acidosis decreased renal TRPM6 expression, increased Mg2+ excretion, and decreased serum Mg2+ concentration, whereas chronic metabolic alkalosis resulted in the exact opposite effects. In conclusion, these data suggest that regulation of Ca2+ and Mg2+ transport proteins contributes importantly to the effects of acid-base status on renal divalent handling.     

Nutrient Transport in the Mammary Gland: Calcium,Trace Minerals and Water Soluble Vitamins

Milk nutrients are secreted by epithelial cells in the alveoli of the mammary gland by several complex and highly coordinated systems. Many of these nutrients are transported from the blood to the milk via transcellular pathways that involve the concerted activity of transport proteins on the apical and basolateral membranes of mammary epithelial cells. In this review, we focus on transport mechanisms that contribute to the secretion of calcium, trace minerals and water soluble vitamins into milk with particular focus on the role of transporters of the SLC series as well as calcium transport proteins (ion channels and pumps). Numerous members of the SLC family are involved in the regulation of essential nutrients in the milk, such as the divalent metal transporter-1 (SLC11A2), ferroportin-1 (SLC40A1) and the copper transporter CTR1 (SLC31A1). A deeper understanding of the physiology and pathophysiology of these transporters will be of great value for drug discovery and treatment of breast diseases.