Mesenchymal Stem Cells and Psoriasis: Systematic Review.

Mesenchymal Stem Cells (MSCs) are multipotent non-hematopoietic stromal cells found in different body tissues such as bone marrow, adipose tissue, periosteum, Wharton's jelly, umbilical cord, blood, placenta, amniotic fluid, and skin. The biological behavior of MSCs depends mainly on their interaction with the microenvironment in which they are found, whose quality deeply influences the regenerative and immunomodulatory properties of these cells. Several studies confirm the interaction between MSCs and inflammatory microenvironment in the pathogenesis of psoriasis, designating MSCs as an important factor driving psoriasis development. This review aims to describe the most recent evidence on how the inflammatory microenvironment that characterizes psoriasis influences the homeostasis of MSCs and how they can be used to treat the disease.

Mesenchymal Stem Cells Exposed to Persistently High Glucocorticoid Levels Develop Insulin-Resistance and Altered Lipolysis: A Promising In Vitro Model to Study Cushing's Syndrome.

Background: In Cushing's syndrome (CS), chronic glucocorticoid excess (GC) and disrupted circadian rhythm lead to insulin resistance (IR), diabetes mellitus, dyslipidaemia and cardiovascular comorbidities. As undifferentiated, self-renewing progenitors of adipocytes, mesenchymal stem cells (MSCs) may display the detrimental effects of excess GC, thus revealing a promising model to study the molecular mechanisms underlying the metabolic complications of CS. Methods: MSCs isolated from the abdominal skin of healthy subjects were treated thrice daily with GCs according to two different regimens: lower, circadian-decreasing (Lower, Decreasing Exposure, LDE) versus persistently higher doses (Higher, Constant Exposure, HCE), aimed at mimicking either the physiological condition or CS, respectively. Subsequently, MSCs were stimulated with insulin and glucose thrice daily, resembling food uptake and both glucose uptake/GLUT-4 translocation and the expression of LIPE, ATGL, IL-6 and TNF-α genes were analyzed at predefined timepoints over three days. Results: LDE to GCs did not impair glucose uptake by MSCs, whereas HCE significantly decreased glucose uptake by MSCs only when prolonged. Persistent signs of IR occurred after 30 hours of HCE to GCs. Compared to LDE, MSCs experiencing HCE to GCs showed a downregulation of lipolysis-related genes in the acute period, followed by overexpression once IR was established. Conclusions: Preserving circadian GC rhythmicity is crucial to prevent the occurrence of metabolic alterations. Similar to mature adipocytes, MSCs suffer from IR and impaired lipolysis due to chronic GC excess: MSCs could represent a reliable model to track the mechanisms involved in GC-induced IR throughout cellular differentiation.

Gateways for Glutamate Neuroprotection in Parkinson's Disease (PD): Essential Role of EAAT3 and NCX1 Revealed in an In Vitro Model of PD.

Increasing evidence suggests that metabolic alterations may be etiologically linked to neurodegenerative disorders such as Parkinson's disease (PD) and in particular empathizes the possibility of targeting mitochondrial dysfunctions to improve PD progression. Under different pathological conditions (i.e., cardiac and neuronal ischemia/reperfusion injury), we showed that supplementation of energetic substrates like glutamate exerts a protective role by preserving mitochondrial functions and enhancing ATP synthesis through a mechanism involving the Na+-dependent excitatory amino acid transporters (EAATs) and the Na+/Ca2+ exchanger (NCX). In this study, we investigated whether a similar approach aimed at promoting glutamate metabolism would be also beneficial against cell damage in an in vitro PD-like model. In retinoic acid (RA)-differentiated SH-SY5Y cells challenged with α-synuclein (α-syn) plus rotenone (Rot), glutamate significantly improved cell viability by increasing ATP levels, reducing oxidative damage and cytosolic and mitochondrial Ca2+ overload. Glutamate benefits were strikingly lost when either EAAT3 or NCX1 expression was knocked down by RNA silencing. Overall, our results open the possibility of targeting EAAT3/NCX1 functions to limit PD pathology by simultaneously favoring glutamate uptake and metabolic use in dopaminergic neurons.

NCX1 and EAAC1 transporters are involved in the protective action of glutamate in an in vitro Alzheimer's disease-like model.

Increasing evidence suggests that metabolic dysfunctions are at the roots of neurodegenerative disorders such as Alzheimer's disease (AD). In particular, defects in cerebral glucose metabolism, which have been often noted even before the occurrence of clinical symptoms and histopathological lesions, are now regarded as critical contributors to the pathogenesis of AD. Hence, the stimulation of energy metabolism, by enhancing the availability of specific metabolites, might be an alternative way to improve ATP synthesis and to positively affect AD progression. For instance, glutamate may serve as an intermediary metabolite for ATP synthesis through the tricarboxylic acid (TCA) cycle and the oxidative phosphorylation. We have recently shown that two transporters are critical for the anaplerotic use of glutamate: the Na+-dependent Excitatory Amino Acids Carrier 1 (EAAC1) and the Na+-Ca2+ exchanger 1 (NCX1). Therefore, in the present study, we established an AD-like phenotype by perturbing glucose metabolism in both primary rat cortical neurons and retinoic acid (RA)-differentiated SH-SY5Y cells, and we explored the potential of glutamate to halt cell damage by monitoring neurotoxicity, AD markers, ATP synthesis, cytosolic Ca2+ levels and EAAC1/NCX1 functional activities. We found that glutamate significantly increased ATP production and cell survival, reduced the increase of AD biomarkers (amyloid ß protein and the hyperphosphorylated form of tau protein), and recovered the increase of NCX reverse-mode activity. The RNA silencing of either EAAC1 or NCX1 caused the loss of the beneficial effects of glutamate, suggesting the requirement of a functional interplay between these transporters for glutamate-induced protection. Remarkably, our results indicate, as proof-of-principle, that facilitating the use of alternative fuels, like glutamate, may be an effective approach to overcome deficits in glucose utilization and significantly slow down neuronal degenerative process in AD.

Multipurpose Na+ ions mediate excitation and cellular homeostasis: Evolution of the concept of Na+ pumps and Na+/Ca2+ exchangers.

Ionic signalling is the most ancient form of regulation of cellular functions in response to environmental challenges. Signals, mediated by Na+ fluxes and spatio-temporal fluctuations of Na+ concentration in cellular organelles and cellular compartments contribute to the most fundamental cellular processes such as membrane excitability and energy production. At the very core of ionic signalling lies the Na+-K+ ATP-driven pump (or NKA) which creates trans-plasmalemmal ion gradients that sustain ionic fluxes through ion channels and numerous Na+-dependent transporters that maintain cellular and tissue homeostasis. Here we present a brief account of the history of research into NKA, Na+ -dependent transporters and Na+ signalling.

Cracking the code of sodium/calcium exchanger (NCX) gating: Old and new complexities surfacing from the deep web of secondary regulations.

Cell membranes spatially define gradients that drive the complexity of biological signals. To guarantee movements and exchanges of solutes between compartments, membrane transporters negotiate the passages of ions and other important molecules through lipid bilayers. The Na+/Ca2+ exchangers (NCXs) in particular play central roles in balancing Na+ and Ca2+ fluxes across diverse proteolipid borders in all eukaryotic cells, influencing cellular functions and fate by multiple means. To prevent progression from balance to disease, redundant regulatory mechanisms cooperate at multiple levels (transcriptional, translational, and post-translational) and guarantee that the activities of NCXs are finely-tuned to cell homeostatic requirements. When this regulatory network is disturbed by pathological forces, cells may approach the end of life. In this review, we will discuss the main findings, controversies and open questions about regulatory mechanisms that control NCX functions in health and disease.

NCX and EAAT transporters in ischemia: At the crossroad between glutamate metabolism and cell survival.

Energy metabolism impairment is a central event in the pathophysiology of ischemia. The limited availability of glucose and oxygen strongly affects mitochondrial activity, thus leading to ATP depletion. In this setting, the switch to alternative energy sources could ameliorate cells survival by enhancing ATP production, thus representing an attractive strategy for ischemic treatment. In this regard, some studies have recently re-evaluated the metabolic role of glutamate and its potential to promote cell survival under pathological conditions. In the present review, we discuss the ability of glutamate to exert an "energizing role" in cardiac and neuronal models of hypoxia/reoxygenation (H/R) injury, focusing on the Na+/Ca2+ exchanger (NCX) and the Na+-dependent excitatory amino acid transporters (EAATs) as key players in this metabolic pathway.

Excitatory Amino Acid Transporters (EAATs): Glutamate Transport and Beyond.

Na+-dependent excitatory amino acid transporters (EAATs) are the major transport mechanisms for extracellular glutamate removal in the central nervous system (CNS). The primary function assigned to EAATs is the maintenance of low extracellular glutamate levels, thus allowing glutamate to be used as a signaling molecule in the brain and to avoid excitotoxicity. However, glutamate has other recognized functions. For instance, it is a key anaplerotic substrate for the tricarboxylic acid (TCA) cycle, as it can be converted to α-ketoglutarate by transaminases or glutamate dehydrogenase. Furthermore, glutamate is a precursor of the main antioxidant glutathione, which plays a pivotal role in preventing oxidative cell death. Therefore, glutamate signaling/use is at the crossroad of multiple metabolic pathways and accordingly, it can influence a plethora of cell functions, both in health and disease. Here, we provide an overview of the main functions of glutamate and its transport systems, analyzing its role as a neurotransmitter and at the same time, the possible metabolic fates it can undergo in the intracellular milieu. Specifically, the metabolic role of glutamate and the molecular machinery proposed to metabolically support its transport will be further analyzed.

Inorganic Polyphosphate Regulates AMPA and NMDA Receptors and Protects Against Glutamate Excitotoxicity via Activation of P2Y Receptors.

Glutamate is one of the most important neurotransmitters in the process of signal transduction in the CNS. Excessive amounts of this neurotransmitter lead to glutamate excitotoxicity, which is accountable for neuronal death in acute neurological disorders, including stroke and trauma, and in neurodegenerative diseases. Inorganic polyphosphate (PolyP) plays multiple roles in the mammalian brain, including function as a calcium-dependent gliotransmitter mediating communication between astrocytes, while its role in the regulation of neuronal activity is unknown. Here we studied the effect of PolyP on glutamate-induced calcium signal in primary rat neurons in both physiological and pathological conditions. We found that preincubation of primary neurons with PolyP reduced glutamate-induced and AMPA-induced but not the NMDA-induced calcium signal. However, in rat hippocampal acute slices, PolyP reduced ion flux through NMDA and AMPA receptors in native neurons. The effect of PolyP on glutamate and specifically on the AMPA receptors was dependent on the presence of P2Y1 but not of P2X receptor inhibitors and also could be mimicked by P2Y1 agonist 2MeSADP. Preincubation of cortical neurons with PolyP significantly reduced the initial calcium peak as well as the number of neurons with delayed calcium deregulation in response to high concentrations of glutamate and resulted in protection of neurons against glutamate-induced cell death. As a result, activation of P2Y1 receptors by PolyP reduced calcium signal acting through AMPA receptors, thus protecting neurons against glutamate excitotoxicity by reduction of the calcium overload and restoration of mitochondrial function.SIGNIFICANCE STATEMENT One of the oldest polymers in the evolution of living matter is the inorganic polyphosphate (PolyP). It is shown to play a role of gliotransmitter in the brain; however, the role of polyphosphate in neuronal signaling is not clear. Here we demonstrate that inorganic polyphosphate is able to reduce calcium signaling induced by physiological or high concentrations of glutamate. The effect of polyphosphate on glutamate-induced calcium signal in neurons is due to the effect of this polymer on the AMPA receptors. The effect of PolyP on glutamate-induced and AMPA-induced calcium signal is dependent on P2Y receptor antagonist. The ability of PolyP to restrict the glutamate-induced calcium signal lies in the basis of its protection of neurons against glutamate excitotoxicity.

Tumor necrosis factor-related apoptosis-inducing ligand reduces the expression of the neuroprotective Na+ /Ca2+ exchanger isoform NCX3 in human neuroblastoma SH-SY5Y cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a cytokine belonging to the TNF superfamily, is regarded as a mediator of neurotoxicity. The constitutively expressed ion exchanger Na+ /Ca2+ exchanger isoform-3 (NCX3) has been shown to protect neurons from injury. Its expression is induced by nerve growth factor (NGF) through activation of its tyrosine kinase receptor trkA. The latter, in turn, activates downstream kinases, such as extracellular signal-regulated kinase (ERK) and the survival-related kinase protein kinase B (AKT). Here, we verified whether TRAIL could influence the expression of NCX3 via modulation of the NGF/trkA system. Differentiated human neuroblastoma SH-SY5Y cells were incubated with TRAIL and, subsequently, the expression of the NCX3 protein was studied at different times by means of western blot analysis. Then, the expression of the phosphorylated forms of either trkA, ERK or AKT was analyzed at identical intervals. Western blot analysis revealed that the expression of NCX3 protein decreased in a time-dependent fashion in SH-SY5Y cells treated with TRAIL, to reach its minimum at 48 h. On the other hand, p-trkA, p-ERK, and p-AKT expression was increased in cells treated with TRAIL after 6 and 16 h; then it declined to nearly undetectable levels after 48 h. Results indicate that the increase in TRAIL expression occurring during neuronal damage may be responsible of NCX3 down-regulation and weakens its neuroprotective effects. The TRAIL system could thus represent a potential target for treatment of neuronal damage characterized by NCX3 function impairment.

Calcium- and ATP-dependent regulation of Na/Ca exchange function in BHK cells: Comparison of NCX1 and NCX3 exchangers.

Na+/Ca2+ exchangers (NCX) mediate bidirectional Ca2+ fluxes across cell membranes and contribute to Ca2+ homeostasis in many cell types. Exchangers are regulated by gating reactions that depend on Na+ and Ca2+ binding to transport and regulatory sites. A Na+i-dependent inactivation is prominent in all isoforms, whereas Ca2+i-dependent regulation varies among isoforms. Here we characterize new details of NCX operation and describe differences and similarities between NCX3 and NCX1 regulation by intracellular Ca2+ and ATP. To compare isoforms, we employed BHK cells expressing NCX3 or NCX1 constitutively and exchange activity was analysed in whole-cell and excised patch recordings under "zero-trans" conditions (i.e., with only one transported ion species on each side). Using BHK cells with low cytoplasmic Ca2+ buffering, outward (reverse) currents, reflecting Ca2+ influx, are activated by applying extracellular Ca2+ (Cao) in the presence of Na+ on the cytoplasmic side. When firstly activated, peak outward NCX3 currents rapidly decay over seconds and then typically develop a secondary transient peak with slower kinetics, until Cao removal abolishes all outward current. The delayed rise of outward current is the signature of an activating process since peak outward NCX3 currents elicited at subsequent Cao bouts remain stimulated for minutes and slower decline towards a non-zero level during continued Cao application. Secondary transient peaks and current stimulation are suppressed by increasing the intracellular Ca2+ buffer capacity or by replacing cytoplasmic ATP with the analogues AMP-PNP or ATPγS. In BHK cells expressing NCX1, outward currents activated under identical settings decay to a steady-state level during single Cao application and are significantly larger, causing strong and long-lived run down of subsequent outward currents. NCX1 current run down is not prevented by increasing cytoplasmic Ca2+ buffering but secondary transient peaks in the outward current profile can be resolved in the presence of ATP. Finally, inward currents recorded in patches excised from NCX3-expressing cells reveal a proteolysis-sensitive, Ca-dependent inactivation process that is unusual for NCX1 forward activity. Together, our results suggest that NCX function is regulated more richly than appreciated heretofore, possibly including processes that are lost in excised membrane patches.

Correction of Mutant p63 in EEC Syndrome Using siRNA Mediated Allele-Specific Silencing Restores Defective Stem Cell Function.

Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) syndrome is a rare autosomal dominant disease caused by heterozygous mutations in the p63 gene and characterized by limb defects, orofacial clefting, ectodermal dysplasia, and ocular defects. Patients develop progressive total bilateral limbal stem cell deficiency, which eventually results in corneal blindness. Medical and surgical treatments are ineffective and of limited benefit. Oral mucosa epithelial stem cells (OMESCs) represent an alternative source of stem cells capable of regenerating the corneal epithelium and, combined with gene therapy, could provide an attractive therapeutic avenue. OMESCs from EEC patients carrying the most severe p63 mutations (p.R279H and p.R304Q) were characterized and the genetic defect of p.R279H silenced using allele-specific (AS) small interfering RNAs (siRNAs). Systematic screening of locked nucleic acid (LNA)-siRNAs against R279H-p63 allele in (i) stable WT-ΔNp63α-RFP and R279H-ΔNp63α-EGFP cell lines, (ii) transient doubly transfected cell lines, and (iii) p.R279H OMESCs, identified a number of potent siRNA inhibitors for the mutant allele, which had no effect on wild-type p63. In addition, siRNA treatment led to longer acquired life span of mutated stem cells compared to controls, less accelerated stem cell differentiation in vitro, reduced proliferation properties, and effective ability in correcting the epithelial hypoplasia, thus giving rise to full thickness stratified and differentiated epithelia. This study demonstrates the phenotypic correction of mutant stem cells (OMESCs) in EEC syndrome by means of siRNA mediated AS silencing with restoration of function. The application of siRNA, alone or in combination with cell-based therapies, offers a therapeutic strategy for corneal blindness in EEC syndrome. Stem Cells 2016;34:1588-1600.

Glutamate-induced ATP synthesis: relationship between plasma membrane Na+/Ca2+ exchanger and excitatory amino acid transporters in brain and heart cell models.

It is known that glutamate (Glu), the major excitatory amino acid in the central nervous system, can be an essential source for cell energy metabolism. Here we investigated the role of the plasma membrane Na(+)/Ca(2+) exchanger (NCX) and the excitatory amino acid transporters (EAATs) in Glu uptake and recycling mechanisms leading to ATP synthesis. We used different cell lines, such as SH-SY5Y neuroblastoma, C6 glioma and H9c2 as neuronal, glial, and cardiac models, respectively. We first observed that Glu increased ATP production in SH-SY5Y and C6 cells. Pharmacological inhibition of either EAAT or NCX counteracted the Glu-induced ATP synthesis. Furthermore, Glu induced a plasma membrane depolarization and an intracellular Ca(2+) increase, and both responses were again abolished by EAAT and NCX blockers. In line with the hypothesis of a mutual interplay between the activities of EAAT and NCX, coimmunoprecipitation studies showed a physical interaction between them. We expanded our studies on EAAT/NCX interplay in the H9c2 cells. H9c2 expresses EAATs but lacks endogenous NCX1 expression. Glu failed to elicit any significant response in terms of ATP synthesis, cell depolarization, and Ca(2+) increase unless a functional NCX1 was introduced in H9c2 cells by stable transfection. Moreover, these responses were counteracted by EAAT and NCX blockers, as observed in SH-SY5Y and C6 cells. Collectively, these data suggest that plasma membrane EAAT and NCX are both involved in Glu-induced ATP synthesis, with NCX playing a pivotal role.

Physical and functional interaction of NCX1 and EAAC1 transporters leading to glutamate-enhanced ATP production in brain mitochondria.

Glutamate is emerging as a major factor stimulating energy production in CNS. Brain mitochondria can utilize this neurotransmitter as respiratory substrate and specific transporters are required to mediate the glutamate entry into the mitochondrial matrix. Glutamate transporters of the Excitatory Amino Acid Transporters (EAATs) family have been previously well characterized on the cell surface of neuronal and glial cells, representing the primary players for glutamate uptake in mammalian brain. Here, by using western blot, confocal microscopy and immunoelectron microscopy, we report for the first time that the Excitatory Amino Acid Carrier 1 (EAAC1), an EAATs member, is expressed in neuronal and glial mitochondria where it participates in glutamate-stimulated ATP production, evaluated by a luciferase-luciferin system. Mitochondrial metabolic response is counteracted when different EAATs pharmacological blockers or selective EAAC1 antisense oligonucleotides were used. Since EAATs are Na(+)-dependent proteins, this raised the possibility that other transporters regulating ion gradients across mitochondrial membrane were required for glutamate response. We describe colocalization, mutual activity dependency, physical interaction between EAAC1 and the sodium/calcium exchanger 1 (NCX1) both in neuronal and glial mitochondria, and that NCX1 is an essential modulator of this glutamate transporter. Only NCX1 activity is crucial for such glutamate-stimulated ATP synthesis, as demonstrated by pharmacological blockade and selective knock-down with antisense oligonucleotides. The EAAC1/NCX1-dependent mitochondrial response to glutamate may be a general and alternative mechanism whereby this neurotransmitter sustains ATP production, since we have documented such metabolic response also in mitochondria isolated from heart. The data reported here disclose a new physiological role for mitochondrial NCX1 as the key player in glutamate-induced energy production.

Clinical pharmacogenetics of methotrexate.

It is well known that interindividual variability can affect the response to many drugs in relation to age, gender, diet, and organ function. Pharmacogenomic studies have also documented that genetic polymorphisms can exert clinically significant effects in terms of drug resistance, efficacy and toxicity by modifying the expression of critical gene products (drug-metabolizing enzymes, transporters, and target molecules) as well as pharmacokinetic and pharmacodynamic parameters. A growing body of in vitro and clinical evidence suggests that common polymorphisms in the folate gene pathway are associated with an altered response to methotrexate (MTX) in patients with malignancy and autoimmune disease. Such polymorphisms may also induce significant MTX toxicity requiring expensive monitoring and treatment. Although the available data are not conclusive, they suggest that in the future MTX pharmacogenetics could play a key role in clinical practice by improving and tailoring treatment. This review describes the genetic polymorphisms that significantly influence MTX resistance, efficacy, and toxicity.

Massive endocytosis driven by lipidic forces originating in the outer plasmalemmal monolayer: a new approach to membrane recycling and lipid domains.

The roles that lipids play in endocytosis are the subject of debate. Using electrical and imaging methods, we describe massive endocytosis (MEND) in baby hamster kidney (BHK) and HEK293 cells when the outer plasma membrane monolayer is perturbed by the nonionic detergents, Triton X-100 (TX100) and NP-40. Some alkane detergents, the amphipathic drugs, edelfosine and tamoxifen, and the phospholipase inhibitor, U73122, are also effective. Uptake of the membrane tracer, FM 4-64, into vesicles and loss of reversible FM 4-64 binding confirm that 40-75% of the cell surface is internalized. Ongoing MEND stops in 2-4 s when amphipaths are removed, and amphipaths are without effect from the cytoplasmic side. Thus, expansion of the outer monolayer is critical. As found for Ca-activated MEND, vesicles formed are <100 nm in diameter, membrane ruffles are lost, and ß-cyclodextrin treatments are inhibitory. However, amphipath-activated MEND does not require Ca transients, adenosine triphosphate (ATP) hydrolysis, G protein cycling, dynamins, or actin cytoskeleton remodeling. With elevated cytoplasmic ATP (>5 mM), MEND can reverse completely and be repeated multiple times in BHK and HEK293 cells, but not cardiac myocytes. Reversal is blocked by N-ethylmaleimide and a nitric oxide donor, nitroprusside. Constitutively expressed Na/Ca exchangers internalize roughly in proportion to surface membrane, whereas Na/K pump activities decrease over-proportionally. Sodium dodecyl sulfate and dodecylglucoside do not cause MEND during their application, but MEND occurs rapidly when they are removed. As monitored capacitively, the binding of these detergents decreases with MEND, whereas TX100 binding does not decrease. In summary, nonionic detergents can fractionate the plasma membrane in vivo, and vesicles formed connect immediately to physiological membrane-trafficking mechanisms. We suggest that lateral and transbilayer inhomogeneities of the plasma membrane provide potential energies that, when unbridled by triggers, can drive endocytosis by lipidic forces.

Massive calcium-activated endocytosis without involvement of classical endocytic proteins.

We describe rapid massive endocytosis (MEND) of >50% of the plasmalemma in baby hamster kidney (BHK) and HEK293 cells in response to large Ca transients. Constitutively expressed Na/Ca exchangers (NCX1) are used to generate Ca transients, whereas capacitance recording and a membrane tracer dye, FM 4-64, are used to monitor endocytosis. With high cytoplasmic adenosine triphosphate (ATP; >5 mM), Ca influx causes exocytosis followed by MEND. Without ATP, Ca transients cause only exocytosis. MEND can then be initiated by pipette perfusion of ATP, and multiple results indicate that ATP acts via phosphatidylinositol-bis 4,5-phosphate (PIP(2)) synthesis: PIP(2) substitutes for ATP to induce MEND. ATP-activated MEND is blocked by an inositol 5-phosphatase and by guanosine 5'-[γ-thio]triphosphate (GTPγS). Block by GTPγS is overcome by the phospholipase C inhibitor, U73122, and PIP(2) induces MEND in the presence of GTPγS. MEND can occur in the absence of ATP and PIP(2) when cytoplasmic free Ca is clamped to 10 µM or more by Ca-buffered solutions. ATP-independent MEND occurs within seconds during Ca transients when cytoplasmic solutions contain polyamines (e.g., spermidine) or the membrane is enriched in cholesterol. Although PIP(2) and cholesterol can induce MEND minutes after Ca transients have subsided, polyamines must be present during Ca transients. MEND can reverse over minutes in an ATP-dependent fashion. It is blocked by brief ß-methylcyclodextrin treatments, and tests for involvement of clathrin, dynamins, calcineurin, and actin cytoskeleton were negative. Therefore, we turned to the roles of lipids. Bacterial sphingomyelinases (SMases) cause similar MEND responses within seconds, suggesting that ceramide may be important. However, Ca-activated MEND is not blocked by reagents that inhibit SMases. MEND is abolished by the alkylating phospholipase A(2) inhibitor, bromoenol lactone, whereas exocytosis remains robust, and Ca influx causes MEND in cardiac myocytes without preceding exocytosis. Thus, exocytosis is not prerequisite for MEND. From these results and two companion studies, we suggest that Ca promotes the formation of membrane domains that spontaneously vesiculate to the cytoplasmic side.

Massive Ca-induced membrane fusion and phospholipid changes triggered by reverse Na/Ca exchange in BHK fibroblasts.

Baby hamster kidney (BHK) fibroblasts increase their cell capacitance by 25-100% within 5 s upon activating maximal Ca influx via constitutively expressed cardiac Na/Ca exchangers (NCX1). Free Ca, measured with fluo-5N, transiently exceeds 0.2 mM with total Ca influx amounting to approximately 5 mmol/liter cell volume. Capacitance responses are half-maximal when NCX1 promotes a free cytoplasmic Ca of 0.12 mM (Hill coefficient approximately 2). Capacitance can return to baseline in 1-3 min, and responses can be repeated several times. The membrane tracer, FM 4-64, is taken up during recovery and can be released at a subsequent Ca influx episode. Given recent interest in signaling lipids in membrane fusion, we used green fluorescent protein (GFP) fusions with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) and diacylglycerol (DAG) binding domains to analyze phospholipid changes in relation to these responses. PI(4,5)P(2) is rapidly cleaved upon activating Ca influx and recovers within 2 min. However, PI(4,5)P(2) depletion by activation of overexpressed hM1 muscarinic receptors causes only little membrane fusion, and subsequent fusion in response to Ca influx remains massive. Two results suggest that DAG may be generated from sources other than PI(4,5)P in these protocols. First, acylglycerols are generated in response to elevated Ca, even when PI(4,5)P(2) is metabolically depleted. Second, DAG-binding C1A-GFP domains, which are brought to the cell surface by exogenous ligands, translocate rapidly back to the cytoplasm in response to Ca influx. Nevertheless, inhibitors of PLCs and cPLA2, PI(4,5)P(2)-binding peptides, and PLD modification by butanol do not block membrane fusion. The cationic agents, FM 4-64 and heptalysine, bind profusely to the extracellular cell surface during membrane fusion. While this binding might reflect phosphatidylserine (PS) "scrambling" between monolayers, it is unaffected by a PS-binding protein, lactadherin, and by polylysine from the cytoplasmic side. Furthermore, the PS indicator, annexin-V, binds only slowly after fusion. Therefore, we suggest that the luminal surfaces of membrane vesicles that fuse to the plasmalemma may be rather anionic. In summary, our results provide no support for any regulatory or modulatory role of phospholipids in Ca-induced membrane fusion in fibroblasts.

Dual control of cardiac Na+ Ca2+ exchange by PIP(2): analysis of the surface membrane fraction by extracellular cysteine PEGylation.

We describe a new assay to determine the fraction of cardiac Na(+)-Ca(2+) exchangers (NCX1) in the surface membrane of cells (F(surf)). An extracellular NCX1 disulphide bond is rapidly reduced by tris(2-carboxyethyl)phosphine hydrochloride (TCEP), cysteines are 'PEGylated' by alkylation with an impermeable conjugate of maleimide and a 5000 MW polyethylene glycol (MPEG), and F(surf) is quantified from Western blots as the fraction of NCX1 that migrates at a higher molecular weight. F(surf) remains less than 0.1 when NCX1 is expressed via transient transfections. Values of 0.15-0.4 are obtained for cell lines with stable NCX1 expression, 0.3 for neonatal myocytes and 0.6-0.8 for adult hearts. To validate the assay, we analysed an intervention that promotes clathrin-independent endocytosis in fibroblasts. Using BHK cells, removal of extracellular potassium (K(+)) caused yellow fluorescent protein (YFP)-tagged NCX1 to redistribute diffusely into the cytoplasm within 30 min, F(surf) decreased by 35%, and whole-cell exchange currents decreased by > 50%. In both HEK 293 and BHK cell lines, expression of human hPIP5Ibeta kinase significantly decreases F(surf). In BHK cells expressing M1 receptors, a muscarinic agonist (carbachol) causes a 40% decrease of F(surf) in normal media. This decrease is blocked by a high wortmannin concentration (3 mum), suggesting that type III phosphatidylinositol-4-kinase (PI4K) activity is required. As predicted from functional studies, carbachol increases F(surf) when cytoplasmic Ca(2) is increased by removing extracellular Na(+). Phorbol esters are without effect in BHK cells. In intact hearts, interventions that change contractility have no effect within 15 min, but we have identified two long-term changes. First, we analysed the diurnal dependence of F(surf) because messages for cardiac phosphatidylinositol-4-phosphate (PIP) 5-kinases increase during the light phase in entrained mice (i.e. during sleep). Cardiac phosphatidylinositol-(4,5)-bis-phosphate (PIP(2)) levels increase during the light phase and F(surf) decreases in parallel. Second, we analysed effects of aortic banding because NCX1 currents do not mirror the increases of NCX1 message and protein that occur in this model. F(surf) decreases significantly within 10 days, and cardiac PIP and PIP(2) levels are significantly increased. In summary, multiple experimental approaches suggest that PIP(2) synthesis favours NCX1 internalization, that NCX1 internalization is probably clathrin-independent, and that significant changes of NCX1 surface expression occur physiologically and pathologically in intact myocardium.