Nuclear sphingolipids: metabolism and signaling

Sphingolipids are most prominently expressed in the plasma membrane, but recent studies have pointed to important signaling and regulatory roles in the nucleus. The most abundant nuclear sphingolipid is sphingomyelin (SM), which occurs in the nuclear envelope (NE) as well as intranuclear sites. The major metabolic product of SM is ceramide, which is generated by nuclear sphingomyelinase and triggers apoptosis and other metabolic changes. Ceramide is further hydrolyzed to free fatty acid and sphingosine, the latter undergoing conversion to sphingosine phosphate by action of a specific nuclear kinase. Gangliosides are another type of sphingolipid found in the nucleus, members of the a-series of gangliotetraose gangliosides (GM1, GD1a) occurring in the NE and endonuclear compartments. GM1 in the inner membrane of the NE is tightly associated with a Na(+)/Ca(2+) exchanger whose activity it potentiates, thereby contributing to regulation of Ca(2+) homeostasis in the nucleus. This was shown to exert a cytoprotective role as absence or inactivation of this nuclear complex rendered cells vulnerable to apoptosis. This was demonstrated in the greatly enhanced kainite-induced seizure activity in knockout mice lacking gangliotetraose gangliosides. The pathology included apoptotic destruction of neurons in the CA3 region of the hippocampus. Ca(2+) homeostasis was restored in these animals with LIGA-20, a membrane-permeant derivative of GM1 that entered the NE and activated the nuclear Na(+)/Ca(2+) exchanger. Some evidence suggests the presence of uncharged glycosphingolipids in the nucleus.     

Sphingolipids of the nucleus and their role in nuclear signaling

Sphingolipids have important signaling and regulatory roles in the nuclei of all vertebrate cells examined to date. Sphingomyelin (SM) is the most abundant of this group and occurs in the nuclear envelope (NE) as well as intranuclear sites. The primary product of SM metabolism is ceramide, whose release by nuclear sphingomyelinase triggers apoptosis and other metabolic changes in the nucleus. Further catabolism results in free fatty acid and sphingosine formation, the latter being capable of conversion to sphingosine phosphate by action of a specific nuclear kinase. Finally, glycosphingolipids such as gangliosides occur in the NE where GM1, one member of the gangliotetraose family, influences Ca(2+) flux by activation of a Na(+)/Ca(2+) exchanger located in the inner membrane of the NE. The tightly associated GM1/exchanger complex was shown to exert a cytoprotective role in neurons and other cell types, as absence of this nuclear complex rendered cells vulnerable to apoptosis. A striking example of this mode of Ca(2+) regulation is the greatly enhanced seizure activity in knockout mice lacking gangliotetraose gangliosides, involving programmed cell death in the CA3 region of the hippocampus. In this model, Ca(2+) homeostasis was restored most effectively with LIGA-20, a membrane-permeant derivative of GM1 that entered the NE and activated the nuclear Na(+)/Ca(2+) exchanger.     

Mutant NG108-15 cells (NG-CR72) deficient in GM1 synthase respond aberrantly to axonogenic stimuli and are vulnerable to calcium-induced apoptosis: they are rescued with LIGA-20

The neuroblastoma x glioma NG108-15 hybrid cell line, a widely used model for the study of neuronal differentiation, contains a variety of gangliosides including GM1 and its sialosylated derivative, GD1a. To investigate the role of these a-series gangliotetraose gangliosides in neuritogenesis, we have obtained a mutated subclone of NG108-15 that is deficient in that family of gangliosides. NG108-15 cells were grown in the presence of cholera toxin, which killed the large majority of cells, and from the cholera-resistant survivors we isolated a clone, NG-CR72, that lacks GM1 and GD1a in the plasma and nuclear membranes. GM2 concentration was significantly higher in the plasma membrane. Enzyme assay indicated deficiency of UDP-Gal:GM2 galactosyltransferase (GM1 synthase), which was confirmed by incorporation studies with [3H]sphingosine. These cells resembled wild-type NG108-15 in extending dendritic processes in response to dendritogenic agents (retinoic acid, dibutyryl cAMP) but responded aberrantly to axonogenic stimuli (KCl, ionomycin) by extending unstable neurites that showed the cytoskeletal staining characteristic of dendrites. Moreover, mutant cells treated with the Ca2+ elevating axonogenic agents underwent apoptosis over time, attributed to dysfunction of Ca2+ regulatory mechanisms normally mediated by GM1. Such agents caused dramatic and sustained elevation of intracellular Ca2+ in mutant cells, in contrast to modest and temporary elevation in wild-type cells. Exogenous GM1, inserted into the plasma membrane, had no discernable protective effect on NG-CR72 cells whereas LIGA-20, a membrane-permeant derivative of GM1 that entered both plasma and nuclear membranes, blocked apoptosis, permitted extension of stable neurites, and attenuated the abnormal elevation of intracellular Ca2+.     

Presence of Sodium–Calcium Exchanger/GM1 Complex in the Nuclear Envelope of Non-neural Cells: Nature of Exchanger-GM1 Interaction

Previous studies have revealed the presence of Na⁺Ca²⁺ exchanger (NCX) activity associated with GM1 ganglioside in the nuclear envelope (NE) of neurons and glia as well as various neural cell lines. The nuclear NCX1 exchanger, unlike that in the plasma membrane, was shown to be tightly associated with GM1 and potentiated by the latter. One non-neural cell line, Jurkat, was found to contain no Na⁺Ca²⁺ exchanger of the NCX1, NCX2, or NCX3 types in either nuclear or plasma membrane. To determine whether such absence in the NE is generally characteristic of non-neural cells we have examined two more such cell lines in addition to human lymphocytes. RT-PCR showed NCX1 expression in both HeLa and NCTC cell lines and also NCX2 in the latter; NCX3, a subtype previously observed in NG108-15 cells, was not expressed in either. Immunocytochemical and immunoblot studies indicated NCX1 on the cell surface and nuclear envelope of both cell types. Some alternatively spliced isoforms of NCX1 in the nuclear envelope of both cell types were tightly associated with ganglioside GM1. Human lymphocytes, a mixed population of T and B cells, showed similar evidence for plasma membrane and nuclear expression in some but not all cells. The high affinity association between NCX1 and GM1, explored by reaction with base, acid, and proteases, was found to involve charge–charge interaction with a requirement for a positively charged moiety in NCX.Special issue dedicated to Lawrence F. Eng.     

Potentiation of a sodium-calcium exchanger in the nuclear envelope by nuclear GM1 ganglioside

Calcium is recognized as an important intracellular messenger with a pivotal role in the regulation of many cytosolic and nuclear processes. Gangliosides of various types, especially GM1, are known to have a role in some aspects of Ca2+ regulation, operating through a variety of mechanisms that are gradually coming to light. The present study provides evidence for a sodium-calcium exchanger in the nuclear envelope of NG108-15 neuroblastoma cells that is potently and specifically activated by GM1. Immunoblot analysis revealed an unusually tight association of GM1 with the exchanger in the nuclear envelope but not with that in the plasma membrane. Exchanger and associated GM1 were located in the inner membrane of the nuclear envelope, suggesting this system could function to transfer Ca2+ between nucleoplasm and the envelope lumen. The GM1-enhanced exchange was blocked by cholera toxin B subunit while C2-ceramide, a recently discovered inhibitor of the exchanger, blocked all transfer. Exchanger activity was significantly elevated in nuclei isolated from cells that were induced to differentiate by KCl + dibutyryl-cAMP, a treatment previously shown to promote up-regulation of nuclear GM1 in conjunction with axonogenesis. Similar enhancement was achieved by addition of exogenous GM1 to nuclei from undifferentiated cells. These results suggest a prominent role for nuclear GM1 in regulation of nuclear Ca2+ homeostasis.     

Susceptibility of cerebellar granule neurons from GM2/GD2 synthase-null mice to apoptosis induced by glutamate excitotoxicity and elevated KCl: Rescue by GM1 and LIGA20

Our previous study showed an impaired regulation of Ca(2+) homeostasis in cultured cerebellar granule neurons (CGN) from neonatal mice lacking GM2, GD2 and all gangliotetraose gangliosides, due to disruption of the GM2/GD2 synthase (GalNAc-T) gene. In the presence of depolarizing concentration (55 mM) K(+), these cells showed persistent elevation of intracellular Ca(2+) ([Ca(2+)]( i )) leading to apoptosis and cell destruction. This was in contrast to CGN from normal littermates whose survival was enhanced by high K(+). In this study we demonstrate that glutamate has the same effect as K(+) on CGN from these ganglioside-deficient knockout (KO) mice and that apoptosis in both cases is averted by exogenous GM1. Even more effective rescue was obtained with LIGA20, a semi-synthetic derivative of GM1. LC(50) of glutamate in the KO cells was 3.1 microM, compared to 46 microM in normal CGN. [Ca(2+)]( i ) measurement with fura-2 revealed no difference in glutamate-stimulated Ca(2+) influx between the 2 cell types. However, reduction of [Ca(2+)]( i ) following application of Mg(2+) was significantly impaired in the mutant CGN. The rescuing effects of exogenous GM1 and LIGA20 corresponded to their ability to restore Ca(2+) homeostasis. The greater potency of LIGA20 is attributed to its greater membrane permeability with resultant ability to insert into both plasma and nuclear membranes at low concentration (相似文献   

GM1 ganglioside: another nuclear lipid that modulates nuclear calcium. GM1 potentiates the nuclear sodium-calcium exchanger

The nuclear envelope (NE) enclosing the cell nucleus, although morphologically and chemically distinct from the plasma membrane, has certain features in common with the latter including the presence of GM1 as an important modulatory molecule. This ganglioside influences Ca(2+) flux across both membranes, but by quite different mechanisms. GM1 in the NE contributes to regulation of nuclear Ca(2+) through potentiation of a Na(+)/Ca(2+) exchanger in the inner nuclear membrane, whereas in the cell membrane, it regulates cytosolic Ca(2+) through modulation of a nonvoltage-gated Ca(2+) channel. Studies with neuroblastoma cells suggest GM1 concentration becomes elevated in the NE with onset of axonogenesis. However, the nuclear GM1/exchanger complex is not limited to neuronal cells but also occurs in NE of astrocytes, C6 cells, and certain non-neural cells. Immunoprecipitation and immunoblot experiments have shown high affinity association of the nuclear Na(+)/Ca(2+) exchanger with GM1, in contrast to Na(+)/Ca(2+) exchangers of the plasma membrane, which bind GM1 less avidly or not at all. This is believed to be due to different isoforms of the exchanger and a difference in topology of GM1 relative to the large inner loop of the exchanger in the 2 membranes. Cultured neurons from mice genetically engineered to lack GM1 suffered Ca(2+) dysregulation as seen in their high vulnerability to Ca(2+)-induced apoptosis. They were rescued by GM1 and more effectively by LIGA20, a membrane-permeant derivative of GM1. The mutant animals were highly susceptible to kainate-induced seizures, which are also a reflection of Ca(2+) dysregulation. The seizures were effectively attenuated by LIGA20 in parallel with the ability of this agent to enter brain cells, insert into the NE, and potentiate Na(+)/Ca(2+) exchange activity in the nucleus. The Na(+)/Ca(2+) exchanger of the NE, in association with nuclear GM1, is thus seen contributing to independent regulation of Ca(2+) by the nucleus in a manner that provides cytoprotection against Ca(2+)-induced apoptosis.     

Sialidase occurs in both membranes of the nuclear envelope and hydrolyzes endogenous GD1a

Previous reports indicated the presence of both gangliosides and sialidase in the nuclear envelope (NE) of primary neurons and the NG108-15 neural cell line. GM1, one of the major gangliosides of this membrane, was shown to be tightly associated with a sodium-calcium exchanger in the inner membrane of the NE and to potentiate exchanger activity. GD1a was the other major ganglioside detected in the NE and, like GM1, occurs in both inner and outer membranes. A subsequent report indicated the presence of sialidase activity in the NE without specification as to which of the two membranes express it. The present study was undertaken to determine the nature and locus of this activity within the NE of two cell lines: NG108-15 and SH-SY5Y. Western blot analysis of the separated membranes revealed occurrence of Neu3 in the inner membrane and Neu1 in the outer membrane of the NE. Moreover, sialidase activity at both sites was shown capable of catalyzing conversion of endogenous GD1a to GM1.     

In Search of a Solution to the Sphinx-Like Riddle of GM1

Among the many glycoconjugates contributing to the sugar code, gangliosides have drawn special attention owing to their predominance as the major sialoglycoconjugate category within the nervous system. However, their occurrence, albeit at lower levels, appears ubiquitous in vertebrate cells and even some invertebrate tissues. Now that over 100 gangliosides have been structurally characterized, their diverse physiological functions constitute a remaining enigma. This has been especially true of GM1, for which a surprising array of functions has already been revealed. Our current research has focused on two areas of GM1 function: (a) signaling induced in neural and immune cells by cross-linking of GM1 in the plasma membrane that leads to activation of TRPC5 (transient receptor potiential, canonical form 5) channels, a process important in neuritogenesis and autoimmune suppression; (b) activation by GM1 of a sodium-calcium exchanger (NCX) in the inner membrane of the nuclear envelope (NE) with resulting modulation of nuclear and cellular calcium. The latter has a role in maintaining neuronal viability, loss of which renders neurons vulnerable to Ca²⁺ overload. Pathological manifestations in mutant mice and their cultured neurons lacking GM1 have shown dramatic rescue with a membrane permeable derivative of GM1 that enters the nucleus and restores NCX activity. Nuclear function of GM1 is related to the presence of neuraminidase in the NE, an enzyme that generates GM1 through hydrolysis of GD1a. A different isoform of this enzyme was found in each of the two membranes of the NE.     

Identification of the sodium-calcium exchanger as the major ricin-binding glycoprotein of bovine rod outer segments and its localization to the plasma membrane

After neuraminidase treatment the Na+/Ca2+ exchanger of bovine rod outer segments was found to specifically bind Ricinus communis agglutinin. SDS gel electrophoresis and Western blotting of ricin-binding proteins purified from rod outer segment membranes by lectin affinity chromatography revealed the existence of two major polypeptides of Mr 215K and 103K, the former of which was found to specifically react with PMe 1B3, a monoclonal antibody specific for the 230-kDa non-neuraminidase-treated Na+/Ca2+ exchanger. Reconstitution of the ricin affinity-purified exchanger into calcium-containing liposomes revealed that neuraminidase treatment had no significant effect on the kinetics of Na+/Ca2+ exchange activation by sodium. We further investigated the density of the Na+/Ca2+ exchanger in disk and plasma membrane preparations using Western blotting, radioimmunoassays, immunoelectron microscopy, and reconstitution procedures. The results indicate that the Na+/Ca2+ exchanger is localized in the rod photoreceptor plasma membrane and is absent or present in extremely low concentrations in disk membranes, as we have previously shown to be the case for the cGMP-gated cation channel. Previous reports describing the existence of Na+/Ca2+ exchange activity in rod outer segment disk membrane preparations may be due to the fusion of plasma membrane components and/or the presence of contaminating plasma membrane vesicles.     

Glutamate Transport and Not Glutamate Receptor Binding Is Stimulated by Gangliosides in a Ca2+-Dependent Manner in Rat Brain Synaptic Plasma Membranes

Crude as well as purified synaptic plasma membrane (SPM) preparations were analyzed for the influence of the ganglioside galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylgluc osyl ceramide (GM1) on high-affinity binding of L-[3H]glutamate. Assayed in two different buffer systems, SPM consistently exhibited increased (40-50%) binding upon incubation with GM1 plus Ca2+, as compared to controls without GM1. Incorporation experiments with 3H-labeled GM1 proved trypsin-stable insertion of GM1 into SPM, with a maximum incorporation of four times the endogenous amount (35 nmol/mg of protein). The observed increase in glutamate binding was not due to a change in the affinity of the binding sites, but to a change in the number of binding sites, and it was absolutely dependent on the presence of Ca2+. A pharmacological profile of the GM1/Ca2+-stimulated glutamate binding is presented. The original classification of the stimulatory effect as an effect on glutamate receptor binding had to be revised to take into account the observed temperature sensitivity of the ganglioside effect, its sensitivity to high osmolarity and to ultrasonication, and the lack of binding stimulation after detergent treatment of membranes or after receptor solubilization. Vesicular space measured in both SPM preparations was found to be around 7 microliters/mg of protein, in ganglioside-treated as well as in control membranes. From the data, it is concluded that a special, Na+- and Cl- -independent form of glutamate transport into resealed membrane vesicles is stimulated by gangliosides in the presence of Ca2+.     

Amiloride and its analogs as tools in the study of ion transport

Amiloride inhibits most plasma membrane Na+ transport systems. We have reviewed the pharmacology of inhibition of these transporters by amiloride and its analogs. Thorough studies of the Na+ channel, the Na+/H+ exchanger, and the Na+/Ca2+ exchanger, clearly show that appropriate modification of the structure of amiloride will generate analogs with increased affinity and specificity for a particular transport system. Introduction of hydrophobic substituents on the terminal nitrogen of the guanidino moiety enhances activity against the Na+ channel; whereas addition of hydrophobic (or hydrophilic) groups on the 5-amino moiety enhances activity against the Na+/H+ exchanger. Activity against the Na+/Ca2+ exchanger and Ca2+ channel is increased with hydrophobic substituents at either of these sites. Appropriate modification of amiloride has produced analogs that are several hundred-fold more active than amiloride against specific transporters. The availability of radioactive and photoactive amiloride analogs, anti-amiloride antibodies, and analogs coupled to support matrices should prove useful in future studies of amiloride-sensitive transport systems. The use of amiloride and its analogs in the study of ion transport requires a knowledge of the pharmacology of inhibition of transport proteins, as well as effects on enzymes, receptors, and other cellular processes, such as DNA, RNA, and protein synthesis, and cellular metabolism. One must consider whether the effects seen on various cellular processes are direct or due to a cascade of events triggered by an effect on an ion transport system.     

Mice Lacking Major Brain Gangliosides Develop Parkinsonism

Parkinson’s disease (PD) is the second most prevalent late-onset neurodegenerative disorder that affects nearly 1% of the global population aged 65 and older. Whereas palliative treatments are in use, the goal of blocking progression of motor and cognitive disability remains unfulfilled. A better understanding of the basic pathophysiological mechanisms underlying PD would help to advance that goal. The present study provides evidence that brain ganglioside abnormality, in particular GM1, may be involved. This is based on use of the genetically altered mice with disrupted gene Galgt1 for GM2/GD2 synthase which depletes GM2/GD2 and all the gangliotetraose gangliosides that constitute the major molecular species of brain. These knockout mice show overt motor disability on aging and clear indications of motor impairment with appropriate testing at an earlier age. This disability was rectified by L-dopa administration. These mice show other characteristic symptoms of PD, including depletion of striatal dopamine (DA), loss of DA neurons of the substantia nigra pars compacta, and aggregation of alpha synuclein. These manifestations of parkinsonism were largely attenuated by administration of LIGA-20, a membrane permeable analog of GM1 that penetrates the blood brain barrier and enters living neurons. These results suggest that perturbation of intracellular mechanisms mediated by intracellular GM1 may be a contributing factor to PD.     

Fluorescence-topographic NSOM directly visualizes peak-valley polarities of GM1/GM3 rafts in cell membrane fluctuations

Simultaneous fluorescence-topographic nanoscale imaging of cell-surface molecules in the context of membrane ultra-structures has not been reported. Here, near-field scanning optical microscopy (NSOM)-based direct fluorescence-topographic imaging indicated that GM3 rafts/nanodomains (190.0 +/- 49.8 nm ranging 84.5-365.0 nm) were localized predominantly on the peaks of microvillus-like protrusions in the apical membrane of GM3 + Madin-Darby canine kidney cells, whereas GM1 rafts/nanodomains (159.5 +/- 63.8 nm ranging 42-360 nm) were distributed mainly on the slops of protrusions or the valleys between protrusions in the plasma membranes of GM1 + MDCK cells. The data demonstrated that gangliosides polarized not only in a well-known apical-basolateral manner but also in the more microscopic peak-valley manner, implicating unique distribution of GM1 or GM3 in cell-surface fluctuations on the apical membrane of polarized cells. The peak-valley polarities of gangliosides also implicated their different functions relevant to lipid rafts, microvilli, or cellular processes. Importantly, our study demonstrated for the first time that the NSOM-based direct fluorescence-topographic imaging is unique and powerful for elucidating nanoscale distribution of specific cell-surface molecules in membrane fluctuations.     

The Effect of Antibodies to Gangliosides on Ca2+ Channel-Linked Release of γ-Aminobutyric Acid in Rat Brain Slices

Antibodies to GM1 ganglioside enhance the release of gamma-aminobutyric acid (GABA) from rat brain slices induced by depolarization with either 40 mM K+ or 200 microM veratrine. Three new observations are now reported. (a) GABA release induced by the Ca2+ ionophore A23187 was not affected by these antibodies. Because this Ca2+ ionophore causes transmitter release by bypassing depolarization-induced opening of Ca2+ channels, this result suggests that gangliosides participate either in the functioning of such Ca2+ channels or in the Na+ channels involved in depolarization. (b) The enhancement (by antibodies to GM1 ganglioside) of GABA release induced by high K+ levels occurred in the presence of tetrodotoxin (0.01 microM). (c) GABA release induced by veratrine in the absence of Ca2+ was not affected by the antibodies. These latter two observations indicate that Na+ channels are not involved in the action of the antibodies. We conclude that this evidence points to the participation of gangliosides in Ca2+ channel functions involved in GABA release in rat brain slices.     

Calcium clearance mechanisms of mouse sperm

The spermatozoon is specialized for a single vital role in fertilization. Past studies show that Ca2+ signals produced by the opening of plasma membrane entry channels initiate several events required for the sperm to reach and enter the egg but reveal little about how resting [Ca2+]i is maintained or restored after elevation. We examined these homeostatic mechanisms by monitoring the kinetics of recovery from depolarizing stimuli under conditions intended to inhibit candidate mechanisms for sequestration or extrusion of Ca2+ from the cytosol. We found that the Ca2+-ATPase pump of the plasma membrane performs the major task of Ca2+ clearance. It is essential in the final stages of recovery to achieve a low resting [Ca2+]i. With immunomethods we found a approximately 130-kD plasma membrane Ca2+-ATPase protein on Western blots of whole sperm extracts and showed immunolocalization to the proximal principal piece of the flagellum. The plasma membrane Na+-Ca2+ exchanger also exports Ca2+ when [Ca2+]i is elevated. Simultaneous inhibition of both mechanisms of extrusion revealed an additional contribution to clearance from a CCCP-sensitive component, presumably sequestration by the mitochondria. Involvement of SERCA pumps was not clearly detected. Many aspects of the kinetics of Ca2+ clearance observed in the presence and absence of inhibitors were reproduced in a mathematical model based on known and assumed kinetic parameters. The model predicts that when cytosolic [Ca2+] is at 1 microM, the rates of removal by the Ca2+-ATPase, Na+-Ca2+-exchanger, mitochondrial uniporter, and SERCA pump are approximately 1.0, 0.35, 0.33, and 0 micromole l(-1) s(-1), rates substantially slower than those reported for other cells studied by similar methods. According to the model, the Na+-Ca2+ exchanger is poised so that it may run in reverse at resting [Ca2+]i levels. We conclude that the essential functions of sperm do not require the ability to recover rapidly from globally elevated cytosolic [Ca2+].     

Vascular Na+/Ca2+ exchanger: implications for the pathogenesis and therapy of salt-dependent hypertension

The Na+/Ca2+ exchanger is an ion transporter that exchanges Na+ and Ca2+ in either Ca2+ efflux or Ca2+ influx mode, depending on membrane potential and transmembrane ion gradients. In arterial smooth muscle cells, the Na+/Ca2+ exchanger is thought to participate in the maintenance of vascular tone by regulating cytosolic Ca2+ concentration. Recent pharmacological and genetic engineering studies have revealed that the Ca2+ influx mode of vascular Na+/Ca2+ exchanger type-1 (NCX1) is involved in the pathogenesis of salt-dependent hypertension. SEA0400, a specific Na+/Ca2+ exchange inhibitor that preferentially blocks the Ca2+ influx mode, lowers arterial blood pressure in salt-dependent hypertensive models, but not in normotensive rats or other types of hypertensive rats. Furthermore, heterozygous mice with reduced expression of NCX1 are resistant to development of salt-dependent hypertension, whereas transgenic mice with vascular smooth muscle-specific overexpression of NCX1 readily develop hypertension after high-salt loading. SEA0400 reverses the cytosolic Ca2+ elevation and vasoconstriction induced by nanomolar ouabain, as well as humoral factors in salt-loaded animals. One possibility is that circulating endogenous cardiotonic steroids may be necessary for NCX1-mediated hypertension. These findings help to explain how arterial smooth muscle cells in blood vessels contribute to salt-elicited blood pressure elevation and suggest that NCX1 inhibitors might be therapeutically useful for salt-dependent hypertension.     

Phosphatidyl inositol-4,5-bisphosphate bound to bovine cardiac Na+/Ca2+ exchanger displays a MgATP regulation similar to that of the exchange fluxes.

This work shows the existence of a phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) bound form of the cardiac sarcolemmal Na+/Ca2+ exchanger. That was demonstrated in Western blots and cross-immunoprecipitation by using specific antibodies against the NCX1 exchanger (NCX1) and against PtdIns-4,5-P2. In addition, PtdIns-4,5-P2 bound to the Na+/Ca2+ exchanger and the Na+/Ca2+ exchange fluxes displayed a similar MgATP regulation: (a) both increase by 100-130% when membrane vesicles are incubated (15-20 s at 37 degrees C) with 1 mM MgATP and 1 microM Ca2+ (b) in the presence of 100 microM Ca2+, MgATP fails to stimulate the exchange fluxes and does not modify the levels of PtdIns-4,5-P2 bound to the exchanger. In addition, in the absence of Ca2+, the net synthesis of total membrane PtdIns-4,5-P2 is greatly reduced compared with that in the presence of 1 microM Ca2+. Furthermore, in the absence of Ca2+ there is no effect of MgATP on the levels of PtdIns-4,5-P2 bound to the exchanger. These results indicate that, in bovine heart, MgATP-stimulation of Na+/Ca2+ exchange is associated with intracellular Ca2+-dependent levels of PtdIns-4,5-P2 bound to the exchanger molecule.     

Fluorescence Polarization Analysis, Lipid Composition, and Na+, K+-ATPase Kinetics of Synaptosomal Membranes in Feline GM1 and GM2 Gangliosidosis

Neurochemical studies were performed on synaptosomal membranes from cats with GM1 or GM2 gangliosidosis to examine possible mechanisms of neuronal dysfunction in these disorders. The basic hypothesis tested was that deficient ganglioside catabolism causes increased ganglioside content of synaptosomal plasma membrane which in turn disrupts normal function. Fluidity characteristics of synaptosomal membranes were examined using fluorescence polarization. Results showed markedly reduced membrane fluidity in both GM1 and GM2 gangliosidosis. These results were supported by a second study which revealed that isolated synaptosomal membranes of GM1 gangliosidosis cats had a 24-fold increase in total ganglioside content caused predominantly by excess GM1, a 2.3-fold increased cholesterol content, and a 1.4-fold increased phospholipid content. Finally, kinetic analysis of synaptosomal plasma membrane Na+,K+-ATPase from cats with GM1 gangliosidosis showed negligible differences in kinetic parameters compared with controls. Thus, the enzyme appeared protected from the global membrane changes in fluidity and composition. These observations provide evidence for a pathogenetic mechanism of neuronal dysfunction in the gangliosidoses while demonstrating protection of certain vital functional components, such as Na+,K+-ATPase.     

Regulation of cytosolic free calcium in rabbit proximal renal tubules

The relative role of various Ca2+ transport systems in the regulation of Ca2+ cytosolic free Ca2+ concentration was evaluated using rabbit renal proximal tubules. Intracellular compartmentation was evaluated through Ca2+ releases induced by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), A23187, and ruthenium red (RR) alone and in combination. In a Ca2+-free solution after 1 h of incubation, FCCP released 43 +/- 4%, A23187 54 +/- 3%, and RR 29 +/- 5% of total cellular Ca2+; in addition, RR inhibited the rate of FCCP-induced release, confirming its mitochondrial origin. In 1 mM Ca2+, the releases were 57 +/- 9%, 70 +/- 5%, and 34 +/- 10%, respectively. Therefore, the mitochondrial Ca2+ content is 20-60 nmol/mg of mitochondrial protein, sufficiently large to effectively buffer cell Ca2+. To evaluate the role of the plasma membrane Na:Ca exchanger, 10(-4) M ouabain was added and caused a slight decline in total cell Ca2+ content and no change in ionized Ca2+ measured by the null-point method, suggesting that the plasmalemmal Na+:Ca2+ exchanger does not play an important role in Ca2+ extrusion. Cytosolic free Ca2+ increased when 100 mM sodium was replaced with equimolar choline or tetramethylammonium. However, tetramethylammonium replacement released 55% of the mitochondrial Ca2+ content by increasing mitochondrial Ca2+ efflux without affecting the Ca2+ influx pathway. These results suggest that Na+ replacements in this tissue increase ionized Ca2+ by increasing mitochondrial Ca2+ efflux and not by inhibition of Na+:Ca2+ exchange at the plasma membrane.