Protective Effects of Ca2+ on Cell Membrane Damage by Polygodial in Saccharomyces cerevisiae

We report here the expression of a bacterial sphingomyelinase in mammalian cells as a functionally active form. A chimeric Pseudomonas sphingomyelinase fused with the lysosomal sorting motif of lysosomal acid phosphatase was sorted to lysosomes in mammalian cells. As expected, the chimeric SMase hydrolyzed sphingomyelin in vivo to produce ceramide, part of which was converted to glucosylceramide.     

Phosphatidylinositol-3,5-Bisphosphate is a potent and selective inhibitor of acid sphingomyelinase

Acid sphingomyelinase (A-SMase, EC 3.1.4.12) catalyzes the lysosomal degradation of sphingomyelin to phosphorylcholine and ceramide. Inherited deficiencies of acid sphingomyelinase activity result in various clinical forms of Niemann-Pick disease, which are characterised by massive lysosomal accumulation of sphingomyelin. Sphingomyelin hydrolysis by both, acid sphingomyelinase and membrane-associated neutral sphingomyelinase, plays also an important role in cellular signaling systems regulating proliferation, apoptosis and differentiation. Here, we present a potent and selective novel inhibitor of A-SMase, L-alpha-phosphatidyl-D-myo-inositol-3,5-bisphosphate (PtdIns3,5P2), a naturally occurring substance detected in mammalian, plant and yeast cells. The inhibition constant Ki for the new A-SMase inhibitor PtdIns3,5P2 is 0.53 microM as determined in a micellar assay system with radiolabeled sphingomyelin as substrate and recombinant human A-SMase purified from insect cells. Even at concentrations of up to 50 microM, PtdIns3,5P2 neither decreased plasma membrane-associated, magnesium-dependent neutral sphingomyelinase activity, nor was it an inhibitor of the lysosomal hydrolases beta-hexosaminidase A and acid ceramidase. Other phosphoinositides tested had no or a much weaker effect on acid sphingomyelinase. Different inositol-bisphosphates were studied to elucidate structure-activity relationships for A-SMase inhibition. Our investigations provide an insight into the structural features required for selective, efficient inhibition of acid sphingomyelinase and may also be used as starting point for the development of new potent A-SMase inhibitors optimised for diverse applications.     

Accurate Differentiation of Neuronopathic and Nonneuronopathic Forms of Niemann-Pick Disease by Evaluation of the Effective Residual Lysosomal Sphingomyelinase Activity in Intact Cells

Abstract: Niemann-Pick disease types A and B are two clinical forms of an inherited lysosomal storage disorder characterized by accumulation of sphingomyelin due to deficient activity of the lysosomal enzyme, acid sphingomyelinase. Patients with both types have hepatosplenomegaly, but only those with type A have nervous system involvement leading to death in early infancy. The residual activities of lysosomal sphingomyelinase in types A and B have never been well characterized because of limitations in both in vitro enzymatic assays and loading tests on intact cells. To evaluate the effective level of sphingomyelinase activity, intact, living cultured Epstein-Barr virus-transformed lymphoid cells were incubated with a radiolabeled sphingomyelin that was first associated to human low-density lipoproteins. This lipoprotein-associated sphingomyelin was targeted to lysosomes, thereby permitting selective hydrolysis by the lysosomal sphingomyelinase. Short-term pulse-chase experiments allowed the determination of the initial rates of degradation; in normal cells, the half-time of sphingomyelin degradation averaged 4.5 h. Whereas cells from the severe neuronopathic type A form of Niemann-Pick disease exhibited about 0.15% residual sphingomyelinase activity, cells from patients with the visceral type B form exhibited about 4%, i.e., 27 times more. Cells from heterozygous Niemann-Pick subjects showed about 70% residual activity. These results provide the first approach to measuring the effective activity of a lysosomal enzyme and represent an accurate method for the differential diagnosis of Niemann-Pick disease types A and B. They also support the hypothesis of relationships among the effective in situ residual enzyme activity, the amount of stored substrate, and the severity of the ensuing lysosomal storage disorder.     

Posttranslational regulation of acid sphingomyelinase in niemann-pick type C1 fibroblasts and free cholesterol-enriched chinese hamster ovary cells

Niemann-Pick type C disease is characterized by the accumulation of cholesterol and other lipids within the lysosomal compartment, a process that is often accompanied by a reduction in acid sphingomyelinase activity. These studies demonstrate that a CHO cell mutant (CT-60), which accumulates lysosomal cholesterol because of a defective NP-C1 protein, has approximately 5-10% of the acid sphingomyelinase activity of its parental cell line (25-RA) or wild type (CHO-K1) cells. The cholesterol-induced reduction in acid sphingomyelinase activity can be reproduced in CHO-K1 cells by incubation in the presence of low density lipoprotein (LDL) and progesterone, which impairs the normal egress of LDL-derived cholesterol from the lysosomal compartment. Kinetic analysis of sphingomyelin hydrolysis in cell extracts suggests that the CT60 cells have a reduced amount of functional acid sphingomyelinase as indicated by a 10-fold reduction in the apparent V(max). Western blot analysis using antibodies generated to synthetic peptides corresponding to segments within the carboxyl-terminal region of acid sphingomyelinase demonstrate that both the CT60 and the LDL/progesterone-treated CHO-K1 cells possess near normal levels of acid sphingomyelinase protein. Likewise, Niemann-Pick type C fibroblasts also displayed normal acid sphingomyelinase protein but negligible levels of acid sphingomyelinase activity. These data suggest that cholesterol-induced inhibition is a posttranslational event, perhaps involving cofactor mediated modulation of enzymatic activity or alterations in acid sphingomyelinase protein trafficking and maturation.     

Lysosomal sphingomyelinase is not solicited for apoptosis signaling.

Stress-induced activation of an acidic sphingomyelinase leading to generation of ceramide, an important lipid mediator, has been associated with apoptosis; however, the implication of this hydrolase has been questioned. The present study aimed at re-evaluating the role of this lysosomal enzyme in apoptosis initiated by different apoptotic inducers. The sensitivity of a series of acid sphingomyelinase-deficient cell lines derived from Niemann-Pick disease patients to stress-induced apoptosis was investigated. We have now shown that stress stimuli, such as anthracyclines, ionizing radiation, and Fas ligation trigger similar apoptotic hallmarks in normal and acid sphingomyelinase-deficient cell lines. Retrovirus-mediated gene correction of enzyme deficiency in Niemann-Pick cells does not modify response to apoptosis. Ceramide production is comparable in normal and Niemann-Pick cells, and increased activity of neutral sphingomyelinase is observed. Thus, our findings cast serious doubts that lysosomal sphingomyelinase activation is responsible for stress-induced apoptosis of cultured cells.     

Plasma Membrane Repair Is Regulated Extracellularly by Proteases Released from Lysosomes

Eukaryotic cells rapidly repair wounds on their plasma membrane. Resealing is Ca^2+-dependent, and involves exocytosis of lysosomes followed by massive endocytosis. Extracellular activity of the lysosomal enzyme acid sphingomyelinase was previously shown to promote endocytosis and wound removal. However, whether lysosomal proteases released during cell injury participate in resealing is unknown. Here we show that lysosomal proteases regulate plasma membrane repair. Extracellular proteolysis is detected shortly after cell wounding, and inhibition of this process blocks repair. Conversely, surface protein degradation facilitates plasma membrane resealing. The abundant lysosomal cysteine proteases cathepsin B and L, known to proteolytically remodel the extracellular matrix, are rapidly released upon cell injury and are required for efficient plasma membrane repair. In contrast, inhibition of aspartyl proteases or RNAi-mediated silencing of the lysosomal aspartyl protease cathepsin D enhances resealing, an effect associated with the accumulation of active acid sphingomyelinase on the cell surface. Thus, secreted lysosomal cysteine proteases may promote repair by facilitating membrane access of lysosomal acid sphingomyelinase, which promotes wound removal and is subsequently downregulated extracellularly by a process involving cathepsin D.     

Connecting Hsp70, sphingolipid metabolism and lysosomal stability

Heat shock protein 70 (Hsp70) is an evolutionary highly conserved molecular chaperone. Upon cancer-associated translocation to the lysosomal compartment, it promotes cell survival by inhibiting lysosomal membrane permeabilization, a hallmark of stress-induced death. We have recently shown that Hsp70 stabilizes lysosomes by binding to the endo-lysosomal lipid bis(monoacylglycero)phosphate (BMP), an essential co-factor for lysosomal sphingolipid catabolism. The Hsp70–BMP interaction enhances the activity of acid sphingomyelinase, an important enzyme that hydrolyzes sphingomyelin. Importantly, treatment with recombinant Hsp70 effectively reverts the dramatic increase in lysosomal volume and decrease in lysosomal stability in cells from patients with Niemann-Pick disease, a genetic disorder associated with reduced acid sphingomyelinase activity. These findings give new insight into the mechanisms controlling lysosomal stability and integrity, and open new exciting possibilities for the treatment of cancer as well as Niemann-Pick disease.     

The role of sphingomyelin in phosphatidylcholine metabolism in cultured human fibroblasts from control and sphingomyelin lipidosis patients and in Chinese hamster ovary cells.

Human fibroblasts in culture take up exogenous [choline-Me-3H,32P]sphingomyelin (SM) from the medium and incorporate it into cellular SM and phosphatidylcholine [Spence, Clarke & Cook (1983) J. Biol. Chem. 258, 8595-8600]. The ratio of [3H]choline/[32P]Pi is similar in SM and phosphatidylcholine, indicating that the phosphocholine (P-Cho) moiety is transferred intact. Similar results are obtained with Niemann-Pick (NP) cells which are deficient in lysosomal sphingomyelinase activity, suggesting that the P-Cho transfer may not be mediated by the lysosomal sphingomyelinase and that alternative pathways of sphingomyelin catabolism are present in cultured cells. In this study we have shown that: (1) the P-Cho pool in control and NP cells incubated with exogenous labelled SM has a specific radioactivity intermediate between that of SM and PtdCho; (2) expansion of the intracellular P-Cho pool by incubation with exogenous choline reduces the incorporation of [3H]choline from SM into PtdCho; and (3) incorporation of P-Cho from SM into PtdCho is decreased at the non-permissive temperature in Chinese hamster ovary cells with a temperature-sensitive mutation in the cytidylyltransferase reaction. These results suggest that incorporation of P-Cho from SM into PtdCho involves a reaction sequence in which P-Cho is hydrolysed from SM by a sphingomyelinase, followed by incorporation of P-Cho into PtdCho via the cytidine pathway of biosynthesis (SM----P-Cho----CDP-Cho----PtdCho). The appreciable incorporation of P-Cho from SM into PtdCho in sphingomyelinase-deficient NP cells suggests a more substantial or effective lysosomal sphingomyelinase activity in intact cells than is measured in vitro, and/or a significant contribution by other sphingomyelinase activities in these cells.     

Lysosomal involvement in cellular turnover of plasma membrane sphingomyelin

At least two isoenzymes of sphingomyelinase (sphingomyelin cholinephosphohydrolase, EC 3.1.4.12), including lysosomal acid sphingomyelinase and nonlysosomal magnesium-dependent neutral sphingomyelinase, catalyse the degradation of sphingomyelin in cultured human skin fibroblasts. A genetically determined disorder of sphingomyelin metabolism, type A Niemann-Pick disease, is characterized by a deficiency of lysosomal acid sphingomyelinase. To investigate the involvement of lysosomes in the degradation of cellular membrane sphingomyelin, we have undertaken studies to compare the turnover of plasma membrane sphingomyelin in fibroblasts from a patient with type A Niemann-Pick disease, which completely lack acid sphingomyelinase activity but retain nonlysosomal neutral sphingomyelinase activity, with turnover in fibroblasts from normal individuals. Plasma membrane sphingomyelin was labeled by incubating cells at low temperature with phosphatidylcholine vesicles containing radioactive sphingomyelin. A fluorescent analog of sphingomyelin, N-4-nitrobenzo-2-oxa-1,3-diazoleaminocaproyl sphingosylphosphorylcholine (NBD-sphingomyelin) is seen to be readily transferred at low temperature from phosphatidylcholine liposomes to the plasma membranes of cultured human fibroblasts. Moreover, when kinetic studies were done in parallel, a constant ratio of [14C]oleoylsphingosylphosphorylcholine ( [14C]sphingomyelin) to NBD-sphingomyelin was taken up at low temperature by the fibroblast cells, suggesting that [14C]sphingomyelin undergoes a similar transfer. The comparison of sphingomyelin turnover at 37 degrees C in normal fibroblasts compared to Niemann-Pick diseased fibroblasts shows that a rapid turnover of plasma membrane-associated sphingomyelin within the first 30 min appears to be similar in both normal and Niemann-Pick diseased cells. This rapid turnover appears to be primarily due to rapid removal of the [14C]sphingomyelin from the cell surface into the incubation medium. During long-term incubation, an increase in the formation of [14C]ceramide correlating with the degradation of [14C]sphingomyelin is observed in normal fibroblasts. In contrast, the level of [14C]ceramide remains constant in Niemann-Pick diseased cells, which correlates with a higher level of intact [14C]sphingomyelin remaining in these cells compared to normal cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca²⁺ is a key signaling event that triggers the repair of mechanical wounds on the plasma membrane within ~30 sec. Recent studies revealed that mammalian cells also reseal their plasma membrane after permeabilization with pore forming toxins in a Ca²⁺-dependent process that involves exocytosis of the lysosomal enzyme acid sphingomyelinase followed by pore endocytosis. Here, we describe the methodology used to demonstrate that the resealing of cells permeabilized by the toxin streptolysin O is also rapid and dependent on Ca²⁺ influx. The assay design allows synchronization of the injury event and a precise kinetic measurement of the ability of cells to restore plasma membrane integrity by imaging and quantifying the extent by which the liphophilic dye FM1-43 reaches intracellular membranes. This live assay also allows a sensitive assessment of the ability of exogenously added soluble factors such as sphingomyelinase to inhibit FM1-43 influx, reflecting the ability of cells to repair their plasma membrane. This assay allowed us to show for the first time that sphingomyelinase acts downstream of Ca²⁺-dependent exocytosis, since extracellular addition of the enzyme promotes resealing of cells permeabilized in the absence of Ca²⁺.     

High sphingomyelin levels induce lysosomal damage and autophagy dysfunction in Niemann Pick disease type A

Niemann Pick disease type A (NPA), which is caused by loss of function mutations in the acid sphingomyelinase (ASM) gene, is a lysosomal storage disorder leading to neurodegeneration. Yet, lysosomal dysfunction and its consequences in the disease are poorly characterized. Here we show that undegraded molecules build up in neurons of acid sphingomyelinase knockout mice and in fibroblasts from NPA patients in which autophagolysosomes accumulate. The latter is not due to alterations in autophagy initiation or autophagosome–lysosome fusion but because of inefficient autophago–lysosomal clearance. This, in turn, can be explained by lysosomal membrane permeabilization leading to cytosolic release of Cathepsin B. High sphingomyelin (SM) levels account for these effects as they can be induced in control cells on addition of the lipid and reverted on SM-lowering strategies in ASM-deficient cells. These results unveil a relevant role for SM in autophagy modulation and characterize autophagy anomalies in NPA, opening new perspectives for therapeutic interventions.     

The yeast Vps10p cytoplasmic tail mediates lysosomal sorting in mammalian cells and interacts with human GGAs.

Yeast Vps10p is a receptor for transport of the soluble vacuolar hydrolase carboxypeptidase Y to the lysosome-like vacuole. Its functional equivalents in mammalian cells are the mannose 6-phosphate receptors that mediate sorting to lysosomes of mannose 6-phosphate-containing lysosomal proteins. A chimeric receptor was constructed by substituting the cytoplasmic domain of M(r) 300,000 mannose 6-phosphate receptor with the Vps10p cytoplasmic tail. Expression of the chimera in cells lacking endogenous mannose 6-phosphate receptors resulted in a subcellular receptor distribution and an efficiency in sorting of lysosomal enzymes similar to that of the wild type M(r) 300,000 mannose 6-phosphate receptor. Moreover, the cytoplasmic tail of the Vps10p was found to interact with GGA1 and GGA2, two mammalian members of a recently discovered family of clathrin-binding cytosolic proteins that participate in trans-Golgi network-endosome trafficking in both mammals and yeast. Our findings suggest a conserved machinery for Golgi-endosome/vacuole sorting and may serve as a model for future studies of yeast proteins.     

Reduction of acid sphingomyelinase activity in human fibroblasts induced by AY-9944 and other cationic amphiphilic drugs

AY-9944 (trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane dihydrochloride), a cationic amphiphilic drug, caused a rapid, irreversible and dose-dependent reduction of acid sphingomyelinase activity in normal human fibroblasts without changing the activities of other lysosomal hydrolases tested. Examinations of activities against synthetic substrates and of the pH-dependency of sphingomyelinase in the drug-treated cells also suggested that the reduction of activity was specific to acid sphingomyelinase. Such a specific reduction was also found with 12 other cationic amphiphilic drugs, most of which have been shown to be inducers of experimental phospholipidosis in animals and/or cultured cells. These results strongly suggest that acid sphingomyelinase is involved in the process of drug-induced lipidosis. The reduction of acid sphingomyelinase seemed not to be due to direct inhibition by these drugs, a specific loss of the enzyme into the culture medium, the presence of inhibitor in the drug-treated cells, or impaired synthesis of the enzyme. There was no indication that changes in the catalytic properties of the enzyme, or changes in the requirement of detergents for its activity occurred in the cell. These results suggest that AY-9944 and other cationic amphiphilic drugs may cause the reduction of acid sphingomyelinase activity by inducing an increased rate of degradation of the enzyme or by causing an irreversible inactivation via some undetected factor.     

Different pathways of uptake and degradation of sphingomyelin by lymphoblastoid cells and the potential participation of the neutral sphingomyelinase.

The metabolism of sphingomyelin (SPM) was investigated in Epstein-Barr virus-transformed lymphoid cell lines from normal individuals and from patients with Niemann-Pick disease Type A (deficient in the acid, lysosomal sphingomyelinase) and familial hypercholesterolemia (lacking the low density lipoprotein receptor). Cells were incubated with the following radioactive or fluorescent SPMs: [choline-methyl-14C] SPM, [oleoyl-3H]SPM, pyrene-propenoyl-SPM (P3:1-SPM), pyrene-butanoyl-SPM (P4-SPM), pyrene-dodecanoyl-SPM (P12-SPM), and pyrene-sulfonylamino-undecanoyl-SPM (PSA11-SPM). Several pathways of uptake and subsequent metabolism of SPM in the lymphoblastoid cells were identified. [choline-methyl-14C]SPM and the P12-analog, administered to the cells in the presence of lipoproteins, were taken up through the apoB/E receptor-dependent pathway of endocytosis and degraded solely by the lysosomal sphingomyelinase. Under similar conditions, the other sphingomyelins, i.e. [oleoyl-3H]SPM, P3:1-SPM, P4-SPM, and PSA11-SPM, were taken up by a low density lipoprotein receptor-independent pathway and degraded mostly by a nonlysosomal sphingomyelinase which also catalyzed their hydrolysis in Niemann-Pick cells. In the absence of serum, all sphingomyelins were taken up by an apoB/E receptor-independent pathway and hydrolyzed by a nonlysosomal sphingomyelinase. Indeed, in vitro assays demonstrated the presence, in lymphoblastoid cells, of the neutral magnesium-activated sphingomyelinase, which was also fully active in the Niemann-Pick cells. In conclusion, our observations are consistent with: (i) the existence in lymphoblastoid cells of several pathways for the uptake and subsequent utilization of SPM; (ii) a major role of lipoproteins for the metabolic routing of the SPM; and (iii) the effect of the structure of the fatty acyl residue of SPM on its possible association with lipoproteins and/or cell membranes.     

Niemann-Pick disease type B: prenatal diagnosis and enzymatic and chemical studies on fetal brain and liver.

Patients with Niemann-Pick disease type A have a severe neurovisceral disease caused by a deficiency of lysosomal sphingomyelinase activity in all tissues examined. The patients with the type B form have signs and symptoms related to storage of sphingomyelin in the spleen, liver, and lungs, while neurologically they remain normal. They also have a severe deficiency of lysosomal sphingomyelinase activity in all tissues previously examined. Here the brain and liver of a fetus with Niemann-Pick disease type B are examined for enzymatic anc chemical changes. Despite careful analysis, no measurable lysosomal sphingomyelinase could be measured in either organ. Lipid changes were comparable to those observed in fetuses aborted with Niemann-Pick disease type A. The affected child in this family is now age 3 and remains neurologically normal but continues to show organ enlargement and lung infiltration of lipids. It appears that the lack of neurological involvement in type B patients cannot be due to an obvious presence of significant lysosomal sphingomyelinase activity in brain.     

The subcellular localization of neutral sphingomyelinase in rat liver.

The subcellular distribution of neutral sphingomyelinase activity has been determined in rat liver. Neutral sphingomyelinase is present in the plasma membrane. This enzyme requires either Mg2+ or Mn2+ for full activity; these cations cannot be replaced by Co2+ or Ca2+. The plasma membrane sphingomyelinase is strongly inhibited by Hg2+. A small amount of neutral spingomyelinase activity appears to be present in microsomes. No neutral sphingomyelinase activity is present in liver mitochondria or bytosol. Lysosomal sphingomyelinase is fully active at pH 4.4--4.8 without added divalent cations. However, between pH 5.0 and 7.5 lysosomal sphingomyelinase activity is stimulated by Mg2+, Mn2+, Co2+, and Ca2+. Below pH 4.8, Mg2+ inhibits the reaction. In contrast to the results obtained with the neutral sphingomyelinase activity of plasma membranes and microsomes, lysosomal sphingomyelinase is unaffected by sulfhydryl inhibitors.     

Human placental sphingomyelinase. Purification to homogeneity, antigenic properties and partial amino-acid sequences of the enzyme

Sphingomyelinase from human placenta was purified to homogeneity in five steps: concanavalin A Sepharose, butyl agarose. Blue Sepharose, sphingosylphosphocholine Sepharose chromatography and FPLC-Mono Q. This lysosomal enzyme has a pH optimum around pH 5.0-6.0. It is a glycoprotein with an approximate molecular mass of 70 kDa which is reduced to 60 kDa by enzymatic deglycosylation. Monospecific antibodies against sphingomyelinase were isolated using sphingomyelinase covalently linked to Sepharose as affinity matrix. These antibodies effectively inhibit the sphingomyelinase activity. Peptides were released from sphingomyelinase by cyanogen bromide or proteolytically by trypsin, proteinase V8 and Lys C for gas phase sequencing. Amino-acid sequences are reported which proved to be the prerequisite for antibody and oligonucleotide screening of the respective human placenta cDNA libraries for the determination of the complete amino acid sequence of human lysosomal sphingomyelinase. In situ hybridisation with a labelled antisense RNA synthesized in vitro using cloned sphingomyelinase-specific cDNA as template, which encodes the peptide sequences described here, revealed the strong expression of sphingomyelinase in human placental villi and normal fibroblasts. Fibroblasts of a Niemann-Pick patient, however, were free of mRNA expressing the sphingomyelinase described here.     

Effects of dimethylsulfoxide on sphingomyelinase activities in normal and Niemann-Pick type A, B and C fibroblasts

The effects of dimethylsulfoxide (DMSO) on sphingomyelinase activity measured at pH range 3.5-8.0 were examined in normal and Niemann-Pick disease type A, B and C fibroblasts culture. In normal cells, a minor activity was observed at pH 7.5, which was 3- to 4-fold lower than a major one at pH 5.0. Both activities at pH 5.0 and 7.5 were Mg2+-independent and localized to lysosomes. Niemann-Pick type C cells had 30-50% residual sphingomyelinase activity at both pH 5.0 and 7.5, as compared to normal control cells, whereas type A and B cells exhibited virtually no activity over the entire pH range examined. Treatment with 2% DMSO caused a marked increase in sphingomyelinase activities at pH 5.0 and 7.5 in normal and Niemann-Pick disease type C cells, while in type A and B cells, both activities remained virtually unchanged after DMSO treatment. The increase in sphingomyelinase activity at pH 5.0 induced in normal cells by DMSO resulted in an increase in the Vmax without a substantial change in the Km and was inhibited by the simultaneous addition of 10 micrograms/ml of cycloheximide. By comparison, a less than 2-fold increase in other lysosomal hydrolase activities was observed after DMSO treatment in all cell lines examined.     

Modifications of sphingomyelin and phosphatidylcholine metabolism by tricyclic antidepressants and phenothiazines

Phenothiazines and tricyclic antidepressants, when added to culture medium, gave rise in several types of cells (C6 rat glioma cells and human fibroblasts), to a decrease in lysosomal sphingomyelinase activity. The effect of chlorpromazine and desipramine was dose dependent, and was observed after 3 hours of incubation with the drugs at concentrations ranging between 1 and 10 microM. In C6 glioma cell cultures, the decrease in sphingomyelinase activity was related to the clinical effectiveness of phenothiazines, tricyclic antidepressants and derivatives. Incorporation of (choline-14C) sphingomyelin showed that the metabolic pathway implying the synthesis of phosphatidylcholine from the hydrolysis of sphingomyelin and/or transfer of phosphorylcholine to phosphatidylcholine was also partially reduced.     

Calmodulin Antagonist W-7 Inhibits Lysosomal Sphingomyelinase Activity in C6 Glioma Cells

N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) is known to be a potent calmodulin antagonist and inhibitor of calmodulin-dependent protein kinases. W-7 and 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine (H-7) are inhibitors of protein kinase C and cyclic nucleotide-dependent protein kinases. In C6 glioma cells, W-7 and not H-7 inhibited dose-dependently acid sphingomyelinase, a result indicating the modulation of this lysosomal enzyme by a calmodulin-dependent system. Other lysosomal enzymes, such as beta-glucosidase, alpha-galactosidase, and arylsulfatase A, were unaffected by W-7 and H-7, a finding indicating a selective effect of W-7 on sphingomyelinase.