The SLC10 family of sodium/bile salt cotransporters contains over 50 members in animal, plant and bacterial species. In man, two well-characterized members and three orphan transporters are known. The Na+/taurocholate cotransporting polypeptide (NTCP; SLC10A1) and the apical sodium-dependent bile salt transporter (ASBT; SLC10A2) are critical components of the enterohepatic circulation of bile salts. NTCP and ASBT are cotransporters that mediate sodium-dependent, electrogenic uptake of mainly bile salts into hepatocytes (NTCP), biliary epithelial cells, ileal enterocytes and renal proximal tubular cells (ASBT).
The aldosterone-sensitive distal nephron (ASDN) includes the late distal convoluted tubule 2, the connecting tubule (CNT) and the collecting duct. The appropriate regulation of sodium (Na+) absorption in the ASDN is essential to precisely match urinary Na+ excretion to dietary Na+ intake whilst taking extra-renal Na+ losses into account. There is increasing evidence that Na+ transport in the CNT is of particular importance for the maintenance of body Na+ balance and for the long-term control of extra-cellular fluid volume and arterial blood pressure. Na+ transport in the CNT critically depends on the activity and abundance of the amiloride-sensitive epithelial sodium channel (ENaC) in the luminal membrane of the CNT cells. As a rate-limiting step for transepithelial Na+ transport, ENaC is the main target of hormones (e.g. aldosterone, angiotensin II, vasopressin and insulin/insulin-like growth factor 1) to adjust transepithelial Na+ transport in this tubular segment. In this review, we highlight the structural and functional properties of the CNT that contribute to the high Na+ transport capacity of this segment. Moreover, we discuss some aspects of the complex pathways and molecular mechanisms involved in ENaC regulation by hormones, kinases, proteases and associated proteins that control its function. Whilst cultured cells and heterologous expression systems have greatly advanced our knowledge about some of these regulatory mechanisms, future studies will have to determine the relative importance of the various pathways in the native tubule and in particular in the CNT.
The H+ -coupled divalent metal-ion transporter DMT1 serves as both the primary entry point for iron into the body (intestinal brush-border uptake) and the route by which transferrin-associated iron is mobilized from endosomes to cytosol in erythroid precursors and other cells. Elucidating the molecular mechanisms of DMT1 will therefore increase our understanding of iron metabolism and the etiology of iron overload disorders. We expressed wild type and mutant DMT1 in Xenopus oocytes and monitored metal-ion uptake, currents and intracellular pH. DMT1 was activated in the presence of an inwardly directed H+ electrochemical gradient. At low extracellular pH (pHo), H+ binding preceded binding of Fe2+ and its simultaneous translocation. However, DMT1 did not behave like a typical ion-coupled transporter at higher pHo, and at pHo 7.4 we observed Fe2+ transport that was not associated with H+ influx. His272 → Ala substitution uncoupled the Fe2+ and H+ fluxes. At low pHo, H272A mediated H+ uniport that was inhibited by Fe2+. Meanwhile H272A-mediated Fe2+ transport was independent of pHo. Our data indicate (i) that H+ coupling in DMT1 serves to increase affinity for Fe2+ and provide a thermodynamic driving force for Fe2+ transport and (ii) that His-272 is critical in transducing the effects of H+ coupling. Notably, our data also indicate that DMT1 can mediate facilitative Fe2+ transport in the absence of a H+ gradient. Since plasma membrane expression of DMT1 is upregulated in liver of hemochromatosis patients, this H+ -uncoupled facilitative Fe2+ transport via DMT1 can account for the uptake of nontransferrin-bound plasma iron characteristic of iron overload disorders.
Glutamate excitotoxicity is a hypothesis that states excessive glutamate causes neuronal dysfunction and degeneration. As glutamate is a major excitatory neurotransmitter in the central nervous system (CNS), the implications of glutamate excitotoxicity are many and far-reaching. Acute CNS insults such as ischaemia and traumatic brain injury have traditionally been the focus of excitotoxicity research. However, glutamate excitotoxicity has also been linked to chronic neurodegenerative disorders such as amyotrophic lateral sclerosis, multiple sclerosis, Parkinson’s disease and others. Despite the continued research into the mechanisms of excitotoxicity, there are currently no pharmacological interventions capable of providing significant neuroprotection in the clinical setting of brain ischaemia or injury. This review addresses the current state of excitotoxic research, focusing on the structure and physiology of glutamate receptors; molecular mechanisms underlying excitotoxic cell death pathways and their interactions with each other; the evidence for glutamate excitotoxicity in acute neurologic diseases; laboratory and clinical attempts at modulating excitotoxicity; and emerging targets for excitotoxicity research.
The SLC34 family of sodium-driven phosphate cotransporters comprises three members: NaPi-IIa (SLC34A1), NaPi-IIb (SLC34A2), and NaPi-IIc (SLC34A3). These transporters mediate the translocation of divalent inorganic phosphate (HPO4 2−) together with two (NaPi-IIc) or three sodium ions (NaPi-IIa and NaPi-IIb), respectively. Consequently, phosphate transport by NaPi-IIa and NaPi-IIb is electrogenic. NaPi-IIa and NaPi-IIc are predominantly expressed in the brush border membrane of the proximal tubule, whereas NaPi-IIb is found in many more organs including the small intestine, lung, liver, and testis. The abundance and activity of these transporters are mostly regulated by changes in their expression at the cell surface and are determined by interactions with proteins involved in scaffolding, trafficking, or intracellular signaling. All three transporters are highly regulated by factors including dietary phosphate status, hormones like parathyroid hormone, 1,25-OH2 vitamin D3 or FGF23, electrolyte, and acid–base status. The physiological relevance of the three members of the SLC34 family is underlined by rare Mendelian disorders causing phosphaturia, hypophosphatemia, or ectopic organ calcifications.
Endothelium-derived nitric oxide (NO) is a paracrine factor that controls vascular tone, inhibits platelet function, prevents adhesion of leukocytes, and reduces proliferation of the intima. An enhanced inactivation and/or reduced synthesis of NO is seen in conjunction with risk factors for cardiovascular disease. This condition, referred to as endothelial dysfunction, can promote vasospasm, thrombosis, vascular inflammation, and proliferation of vascular smooth muscle cells. Vascular oxidative stress with an increased production of reactive oxygen species (ROS) contributes to mechanisms of vascular dysfunction. Oxidative stress is mainly caused by an imbalance between the activity of endogenous pro-oxidative enzymes (such as NADPH oxidase, xanthine oxidase, or the mitochondrial respiratory chain) and anti-oxidative enzymes (such as superoxide dismutase, glutathione peroxidase, heme oxygenase, thioredoxin peroxidase/peroxiredoxin, catalase, and paraoxonase) in favor of the former. Also, small molecular weight antioxidants may play a role in the defense against oxidative stress. Increased ROS concentrations reduce the amount of bioactive NO by chemical inactivation to form toxic peroxynitrite. Peroxynitrite-in turn-can "uncouple" endothelial NO synthase to become a dysfunctional superoxide-generating enzyme that contributes to vascular oxidative stress. Oxidative stress and endothelial dysfunction can promote atherogenesis. Therapeutically, drugs in clinical use such as ACE inhibitors, AT(1) receptor blockers, and statins have pleiotropic actions that can improve endothelial function. Also, dietary polyphenolic antioxidants can reduce oxidative stress, whereas clinical trials with antioxidant vitamins C and E failed to show an improved cardiovascular outcome.
The Transient Receptor Potential Ankyrin 1 channel (TRPA1), is a member of the large TRP family of ion channels, and functions as a Ca2+ permeable non-selective cation channel in many different cell processes, ranging from sensory to homeostatic tasks. TRPA1 is highly conserved across the animal kingdom. The only mammalian TRPA subfamily member, TRPA1, is widely expressed in neuronal (e.g. sensory dorsal root and trigeminal ganglia neurons)- and in non-neuronal cells (e.g. epithelial cells, hair cells). It exhibits 14–19 amino-(N-)terminal ankyrin repeats, an unusual structural feature. The TRPA1 channel is activated by noxious cold (<17 °C) as well as by a plethora of chemical compounds that includes not only electrophilic compounds and oxidants that can modify, in an alkylative or oxidative fashion, nucleophilic cysteine residues in the channel’s N-terminus, but also compounds that do not covalently bind to the channel proteins (e.g. menthol, nifedipin). Based on localization and functional properties, TRPA1 is considered a key player in acute and chronic (neuropathic) pain and inflammation. Moreover, its role in the (patho)physiology of nearly all organ systems is anticipated, and will be discussed along with the potential of TRPA1 as a drug target for the management of various pathological conditions.
Sleep and the circadian system exert a strong regulatory influence on immune functions. Investigations of the normal sleep–wake cycle showed that immune parameters like numbers of undifferentiated naïve T cells and the production of pro-inflammatory cytokines exhibit peaks during early nocturnal sleep whereas circulating numbers of immune cells with immediate effector functions, like cytotoxic natural killer cells, as well as anti-inflammatory cytokine activity peak during daytime wakefulness. Although it is difficult to entirely dissect the influence of sleep from that of the circadian rhythm, comparisons of the effects of nocturnal sleep with those of 24-h periods of wakefulness suggest that sleep facilitates the extravasation of T cells and their possible redistribution to lymph nodes. Moreover, such studies revealed a selectively enhancing influence of sleep on cytokines promoting the interaction between antigen presenting cells and T helper cells, like interleukin-12. Sleep on the night after experimental vaccinations against hepatitis A produced a strong and persistent increase in the number of antigen-specific Th cells and antibody titres. Together these findings indicate a specific role of sleep in the formation of immunological memory. This role appears to be associated in particular with the stage of slow wave sleep and the accompanying pro-inflammatory endocrine milieu that is hallmarked by high growth hormone and prolactin levels and low cortisol and catecholamine concentrations.
This review aims at presenting state-of-the-art knowledge on the composition and functions of the endothelial glycocalyx. The endothelial glycocalyx is a network of membrane-bound proteoglycans and glycoproteins, covering the endothelium luminally. Both endothelium- and plasma-derived soluble molecules integrate into this mesh. Over the past decade, insight has been gained into the role of the glycocalyx in vascular physiology and pathology, including mechanotransduction, hemostasis, signaling, and blood cell–vessel wall interactions. The contribution of the glycocalyx to diabetes, ischemia/reperfusion, and atherosclerosis is also reviewed. Experimental data from the micro- and macrocirculation alludes at a vasculoprotective role for the glycocalyx. Assessing this possible role of the endothelial glycocalyx requires reliable visualization of this delicate layer, which is a great challenge. An overview is given of the various ways in which the endothelial glycocalyx has been visualized up to now, including first data from two-photon microscopic imaging.
Fluorescent compounds based on synthetic small molecules are powerful tools to visualize biological events in living cells and organisms. Ever since the discovery of organic fluorescent compounds in the late nineteenth century, efforts have been made to “see” the behaviors of specific biomolecules in living systems by using these dyes as labels. Also, following the development of fluorescent Ca2+ indicators in the 1980s, many fluorescent probes or biosensors, which are defined as molecules that show a change in fluorescence properties in the presence of their target molecule, have been reported and applied in biological research. Today, a variety of probes are available that target metal ions, pH, enzyme activities, and signaling molecules. In this review, we first consider the history of organic fluorescent molecules and discuss their utility for labeling biomolecules and staining cells. Then, we review recent progress in small-molecule fluorescent probes for metal ions and reactive oxygen species, focusing on representative work in each category. Finally, we briefly discuss attempts to create novel kinds of probes, including hybrids of small molecules and genetically encoded proteins, with the potential to overcome some of the limitations of current probes.
The term endothelium-derived hyperpolarising factor (EDHF) was introduced in 1987 to describe the hypothetical factor responsible for myocyte hyperpolarisations not associated with nitric oxide (EDRF) or prostacyclin. Two broad categories of EDHF response exist. The classical EDHF pathway is blocked by apamin plus TRAM-34 but not by apamin plus iberiotoxin and is associated with endothelial cell hyperpolarisation. This follows an increase in intracellular [Ca2+] and the opening of endothelial SKCa and IKCa channels preferentially located in caveolae and in endothelial cell projections through the internal elastic lamina, respectively. In some vessels, endothelial hyperpolarisations are transmitted to myocytes through myoendothelial gap junctions without involving any EDHF. In others, the K+ that effluxes through SKCa activates myocytic and endothelial Ba2+-sensitive K-IR channels leading to myocyte hyperpolarisation. K+ effluxing through IKCa activates ouabain-sensitive Na+/K+-ATPases generating further myocyte hyperpolarisation. For the classical pathway, the hyperpolarising "factor" involved is the K+ that effluxes through endothelial K-Ca channels. During vessel contraction, K+ efflux through activated myocyte BKCa channels generates intravascular K+ clouds. These compromise activation of Na+/K+-ATPases and K-IR channels by endothelium-derived K+ and increase the importance of gap junctional electrical coupling in myocyte hyperpolarisations. The second category of EDHF pathway does not require endothelial hyperpolarisation. It involves the endothelial release of factors that include NO, HNO, H2O2 and vasoactive peptides as well as prostacyclin and epoxyeicosatrienoic acids. These hyperpolarise myocytes by opening various populations of myocyte potassium channels, but predominantly BKCa and/or K-ATP, which are sensitive to blockade by iberiotoxin or glibenclamide, respectively.
Novel affinity-purified antibodies against human SGLT1 (hSGLT1) and SGLT2 (hSGLT2) were used to localize hSGLT2 in human kidney and hSGLT1 in human kidney, small intestine, liver, lung, and heart. The renal locations of both transporters largely resembled those in rats and mice; hSGLT2 and SGLT1 were localized to the brush border membrane (BBM) of proximal tubule S1/S2 and S3 segments, respectively. Different to rodents, the renal expression of hSGLT1 was absent in thick ascending limb of Henle (TALH) and macula densa, and the expression of both hSGLTs was sex-independent. In small intestinal enterocytes, hSGLT1 was localized to the BBM and subapical vesicles. Performing double labeling with glucagon-like peptide 1 (GLP-1) or glucose-dependent insulinotropic peptide (GIP), hSGLT1 was localized to GLP-1-secreting L cells and GIP-secreting K cells as has been shown in mice. In liver, hSGLT1 was localized to biliary duct cells as has been shown in rats. In lung, hSGLT1 was localized to alveolar epithelial type 2 cells and to bronchiolar Clara cells. Expression of hSGLT1 in Clara cells was verified by double labeling with the Clara cell secretory protein CC10. Double labeling of human heart with aquaporin 1 immunolocalized the hSGLT1 protein in heart capillaries rather than in previously assumed myocyte sarcolemma. The newly identified locations of hSGLT1 implicate several extra renal functions of this transporter, such as fluid absorption in the lung, energy supply to Clara cells, regulation of enteroendocrine cells secretion, and release of glucose from heart capillaries. These functions may be blocked by reversible SGLT1 inhibitors which are under development.
Endothelial cells situated at the interface between blood and the vessel wall play a crucial role in controlling vascular tone and homeostasis, particularly in determining the expression of pro- and anti-atherosclerotic genes. Many of these effects are mediated by changes in the generation and release of the vasodilator nitric oxide (NO) in response to hemodynamic stimuli exerted on the luminal surface of endothelial cells by the streaming blood (shear stress) and the cyclic strain of the vascular wall. The endothelial NO synthase (eNOS) is activated in response to fluid shear stress and numerous agonists via cellular events such as; increased intracellular Ca2+, interaction with substrate and co-factors, as well as adaptor and regulatory proteins, protein phosphorylation, and through shuttling between distinct sub-cellular domains. Dysregulation of these processes leads to attenuated eNOS activity and reduced NO output which is a characteristic feature of numerous patho-physiological disorders such as diabetes and atherosclerosis. This review summarizes some of the recent findings relating to the molecular events regulating eNOS activity.
The Ca2+ sensor stromal interacting molecule 1 (STIM1) and the Ca2+ channel Orai1 mediate the ubiquitous store-operated Ca2+ entry (SOCE) pathway activated by depletion of internal Ca2+ stores and mediated through the highly Ca2+-selective, Ca2+ release-activated Ca2+ (CRAC) current. Furthermore, STIM1 and Orai1, along with Orai3, encode store-independent Ca2+ currents regulated by either arachidonate or its metabolite, leukotriene C4. Orai channels are emerging as important contributors to numerous cell functions, including proliferation, migration, differentiation, and apoptosis. Recent studies suggest critical involvement of STIM/Orai proteins in controlling the development of several cancers, including malignancies of the breast, prostate, and cervix. Here, we quantitatively compared the magnitude of SOCE and the expression levels of STIM1 and Orai1 in non-malignant human primary astrocytes (HPA) and in primary human cell lines established from surgical samples of the brain tumor glioblastoma multiforme (GBM). Using Ca2+ imaging, patch-clamp electrophysiology, pharmacological reagents, and gene silencing, we established that in GBM cells, SOCE and CRAC are mediated by STIM1 and Orai1. We further found that GBM cells show upregulation of SOCE and increased Orai1 levels compared to HPA. The functional significance of SOCE was evaluated by studying the effects of STIM1 and Orai1 knockdown on cell proliferation and invasion. Utilizing Matrigel assays, we demonstrated that in GBM, but not in HPA, downregulation of STIM1 and Orai1 caused a dramatic decrease in cell invasion. In contrast, the effects of STIM1 and Orai1 knockdown on GBM cell proliferation were marginal. Overall, these results demonstrate that STIM1 and Orai1 encode SOCE and CRAC currents and control invasion of GBM cells. Our work further supports the potential use of channels contributed by Orai isoforms as therapeutic targets in cancer.
Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family of secreted proteins. Signaling cascades induced by BDNF and its receptor, the receptor tyrosine kinase TrkB, link neuronal growth and differentiation with synaptic plasticity. For this reason, interference with BDNF signaling has emerged as a promising strategy for potential treatments in psychiatric and neurological disorders. In many brain circuits, synaptically released BDNF is essential for structural and functional long-term potentiation, two prototypical cellular models of learning and memory formation. Recent studies have revealed an unexpected complexity in the synaptic communication of mature BDNF and its precursor proBDNF, not only between local pre- and postsynaptic neuronal targets but also with participation of glial cells. Here, we consider recent findings on local actions of the BDNF family of ligands at the synapse and discuss converging lines of evidence which emerge from per se conflicting results.
This review examines the role of hormones in the development of social and nonsocial cognition and the brain. Research findings from human studies designed to elucidate the effects of both prenatal and postnatal exposure to hormones in children and young adults are summarized. Effects are found to be both time and dose dependent, with exposure to abnormal hormone levels having a limited impact outside the “critical window” in development. Particular attention is given to the role of prenatal hormone exposure, which appears to be vital for early organization of the brain. In later life, measurements of circulating hormone levels and the administration of testosterone and oxytocin are found to predict behavior, but the effect is thought to be one of “activation” or “fine-tuning” of the early organization of the brain. Possible directions for valuable future research are discussed.
Skeletal muscle atrophy occurs in many chronic diseases and disuse conditions. Its severity reduces patient recovery, independence and quality of life. The discovery of two muscle-specific E3 ubiquitin ligases, MAFbx/atrogin-1 and Muscle RING Finger-1 (MuRF1), promoted an expectation of these molecules as targets for therapeutic development. While numerous studies have determined the conditions in which MAFbx/atrogin-1 and MuRF1 mRNA levels are regulated, few studies have investigated their functional role in skeletal muscle. Recently, studies identifying new target substrates for MAFbx/atrogin-1 and MuRF1, outside of their response to the initiation of muscle atrophy, suggest that there is more to these proteins than previously appreciated. This review will highlight our present knowledge of MAFbx/atrogin-1 and MuRF1 in skeletal muscle atrophy, the impact of potential therapeutics and their known regulators and substrates. Finally, we will comment on new approaches that may expand our knowledge of these two molecules in their control of skeletal muscle function.
Alzheimer's disease (AD) is a progressive neurodegenerative disorder caused by an increase in amyloid metabolism. The calcium hypothesis of AD explores how activation of the amyloidogenic pathway may function to remodel the neuronal Ca2+ signaling pathways responsible for cognition. Hydrolysis of the beta-amyloid precursor protein (APP) yields two products that can influence Ca2+ signaling. Firstly, the amyloids released to the outside form oligomers that enhance the entry of Ca2+ that is pumped into the endoplasmic reticulum (ER). An increase in the luminal level of Ca2+ within the ER enhances the sensitivity of the ryanodine receptors (RYRs) to increase the amount of Ca2+ being released from the internal stores. Secondly, the APP intracellular domain may alter the expression of key signaling components such as the RYR. It is proposed that this remodeling of Ca2+ signaling will result in the learning and memory deficits that occur early during the onset of AD. In particular, the Ca2+ signaling remodeling may erase newly acquired memories by enhancing the mechanism of long-term depression that depends on activation of the Ca2+-dependent protein phosphatase calcineurin. The alteration in Ca2+ signaling will also contribute to the neurodegeneration that characterizes the later stages of dementia.
Aquaporin (AQP) water channels are expressed primarily in cell plasma membranes. In this paper, we review recent evidence that AQPs facilitate cell migration. AQP-dependent cell migration has been found in a variety of cell types in vitro and in mice in vivo. AQP1 deletion reduces endothelial cell migration, limiting tumor angiogenesis and growth. AQP4 deletion slows the migration of reactive astrocytes, impairing glial scarring after brain stab injury. AQP1-expressing tumor cells have enhanced metastatic potential and local infiltration. Impaired cell migration has also been seen in AQP1-deficient proximal tubule epithelial cells, and AQP3-deficient corneal epithelial cells, enterocytes, and skin keratinocytes. The mechanisms by which AQPs enhance cell migration are under investigation. We propose that, as a consequence of actin polymerization/depolymerization and transmembrane ionic fluxes, the cytoplasm adjacent to the leading edge of migrating cells undergoes rapid changes in osmolality. AQPs could thus facilitate osmotic water flow across the plasma membrane in cell protrusions that form during migration. AQP-dependent cell migration has potentially broad implications in angiogenesis, tumor metastasis, wound healing, glial scarring, and other events requiring rapid, directed cell movement. AQP inhibitors may thus have therapeutic potential in modulating these events, such as slowing tumor growth and spread, and reducing glial scarring after injury to allow neuronal regeneration.