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 (PIP2) synthesis: PIP2 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 PIP2 induces MEND in the presence of GTPγS. MEND can occur in the absence of ATP and PIP2 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 PIP2 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 β-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 A2 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.

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 1 /Ca 21 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 21 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 21 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.

Alzheimer's Disease (AD) is a neurodegenerative disorder characterized by progressive neuronal loss. AD is associated with aberrant processing of the amyloid precursor protein, which leads to the deposition of amyloid-β plaques within the brain. Together with plaques deposition, the hyperphosphorylation of the microtubules associated protein tau and the formation of intraneuronal neurofibrillary tangles are a typical neuropathological feature in AD brains. Cellular dysfunctions involving specific subcellular compartments, such as mitochondria and endoplasmic reticulum (ER), are emerging as crucial players in the pathogenesis of AD, as well as increased oxidative stress and dysregulation of calcium homeostasis. Specifically, dysregulation of intracellular calcium homeostasis has been suggested as a common proximal cause of neural dysfunction in AD. Aberrant calcium signaling has been considered a phenomenon mainly related to the dysfunction of intracellular calcium stores, which can occur in both neuronal and nonneuronal cells. This review reports the most recent findings on cellular mechanisms involved in the pathogenesis of AD, with main focus on the control of calcium homeostasis at both cytosolic and mitochondrial level.

Cardiac Na + -Ca 2+ exchange (NCX1) inactivates in excised membrane patches when cytoplasmic Ca 2+ is removed or cytoplasmic Na + is increased. Exogenous phosphatidylinositol-4,5-bisphosphate (PIP 2 ) can ablate both inactivation mechanisms, while it has no effect on inward exchange current in the absence of cytoplasmic Na + . To probe PIP 2 effects in intact cells, we manipulated PIP 2 metabolism by several means. First, we used cell lines with M1 (muscarinic) receptors that couple to phospholipase C's (PLCs). As expected, outward NCX1 current (i.e. Ca 2+ influx) can be strongly inhibited when M1 agonists induce PIP 2 depletion. However, inward currents (i.e. Ca 2+ extrusion) without cytoplasmic Na + can be increased markedly in parallel with an increase of cell capacitance (i.e. membrane area). Similar effects are incurred by cytoplasmic perfusion of GTPγS or the actin cytoskeleton disruptor latrunculin, even in the presence of non-hydrolysable ATP (AMP-PNP). Thus, G-protein signalling may increase NCX1 currents by destabilizing membrane cytoskeleton-PIP 2 interactions. Second, to increase PIP 2 we directly perfused PIP 2 into cells. Outward NCX1 currents increase as expected. But over minutes currents decline substantially, and cell capacitance usually decreases in parallel. Third, using BHK cells with stable NCX1 expression, we increased PIP 2 by transient expression of a phosphatidylinositol-4-phosphate-5-kinase (hPIP5KIβ) and a PI4-kinase (PI4KIIα). NCX1 current densities were decreased by > 80 and 40%, respectively. Fourth, we generated transgenic mice with 10-fold cardiac-specific overexpression of PI4KIIα. This wortmannin-insensitive PI4KIIα was chosen because basal cardiac phosphoinositides are nearly insensitive to wortmannin, and surface membrane PI4-kinase activity, defined functionally in excised patches, is not blocked by wortmannin. Both phosphatidylinositol-4-phosphate (PIP) and PIP 2 were increased significantly, while NCX1 current densities were decreased by 78% with no loss of NCX1 expression. Most mice developed cardiac hypertrophy, and immunohistochemical analysis suggests that NCX1 is redistributed away from the outer sarcolemma. Cholera toxin uptake was increased 3-fold, suggesting that clathrin-independent endocytosis is enhanced. We conclude that direct effects of PIP 2 to activate NCX1 can be strongly modulated by opposing mechanisms in intact cells that probably involve membrane cytoskeleton remodelling and membrane trafficking.

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.

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Baby hamster kidney (BHK) fi broblasts increase their cell capacitance by 25 -100% within 5 s upon activating maximal Ca infl ux via constitutively expressed cardiac Na/Ca exchangers (NCX1). Free Ca, measured with fl uo-5N, transiently exceeds 0.2 mM with total Ca infl ux amounting to ‫ف‬ 5 mmol/liter cell volume. Capacitance responses are half-maximal when NCX1 promotes a free cytoplasmic Ca of 0.12 mM (Hill coeffi cient ≈ 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 infl ux episode. Given recent interest in signaling lipids in membrane fusion, we used green fl uorescent 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 infl ux 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 infl ux 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 infl ux. Nevertheless, inhibitors of PLCs and cPLA2, PI(4,5)P 2 -binding peptides, and PLD modifi cation 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 refl ect 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 fi broblasts.on May 9, 2018 jgp.rupress.org Downloaded from

In this article, the primary institutional affiliation of Drs. Vincenzo Lariccia and Simona Magi should have been given as: