Human vascular endothelial cells are a rich and regulatable source of secretory sphingomyelinase. Implications for early atherogenesis and ceramide-mediated cell signaling

We recently reported that macrophages and fibroblasts secrete a Zn2+-dependent sphingomyelinase (S-SMase), which, like lysosomal SMase, is a product of the acid SMase gene. S-SMase may cause subendothelial retention and aggregation of lipoproteins during atherogenesis, and the acid SMase gene has been implicated in ceramide-mediated cell signaling, especially involving apoptosis of endothelial cells. Because of the central importance of the endothelium in each of these processes, we now sought to examine the secretion and regulation of S-SMase by vascular endothelial cells. Herein we show that cultured human coronary artery and umbilical vein endothelial cells secrete massive amounts of S-SMase (up to 20-fold more than macrophages). Moreover, whereas S-SMase secreted by macrophages and fibroblasts is almost totally dependent on the addition of exogenous Zn2+, endothelium-derived S-SMase was partially active even in the absence of added Zn2+. Secretion of S-SMase by endothelial cells occurred both apically and basolaterally, suggesting an endothelial contribution to both serum and arterial wall SMase. When endothelial cells were incubated with inflammatory cytokines, such as interleukin-1beta and interferon-gamma, S-SMase secretion by endothelial cells was increased 2-3-fold above the already high level of basal secretion, whereas lysosomal SMase activity was decreased. The mechanism of interleukin-1beta-stimulated secretion appears to be through increased routing of a SMase precursor protein through the secretory pathway. In summary, endothelial cells are a rich and regulatable source of enzymatically active S-SMase, suggesting physiologic and pathophysiologic roles for this enzyme.     

Secretory sphingomyelinase, a product of the acid sphingomyelinase gene, can hydrolyze atherogenic lipoproteins at neutral pH. Implications for atherosclerotic lesion development

The subendothelial aggregation and retention of low density lipoprotein (LDL) are key events in atherogenesis, but the mechanisms in vivo are not known. Previous studies have shown that treatment of LDL with bacterial sphingomyelinase (SMase) in vitro leads to the formation of lesion-like LDL aggregates that become retained on extracellular matrix and stimulate macrophage foam cell formation. In addition, aggregated human lesional LDL, but not unaggregated lesional LDL or plasma LDL, shows evidence of hydrolysis by an arterial wall SMase in vivo, and several arterial wall cell types secrete a SMase (S-SMase). S-SMase, however, has a sharp acid pH optimum using a standard in vitro SM-micelle assay. Thus, a critical issue regarding the potential role of S-SMase in atherogenesis is whether the enzyme can hydrolyze lipoprotein-SM, particularly at neutral pH. We now show that S-SMase can hydrolyze and aggregate native plasma LDL at pH 5.5 but not at pH 7.4. Remarkably, LDL modified by oxidation, treatment with phospholipase A2, or enrichment with apolipoprotein CIII, which are modifications associated with increased atherogenesis, is hydrolyzed readily by S-SMase at pH 7.4. In addition, lipoproteins from the plasma of apolipoprotein E knock-out mice, which develop extensive atherosclerosis, are highly susceptible to hydrolysis and aggregation by S-SMase at pH 7.4; a high SM:PC ratio in these lipoproteins appears to be an important factor in their susceptibility to S-SMase. Most importantly, LDL extracted from human atherosclerotic lesions, which is enriched in sphingomyelin compared with plasma LDL, is hydrolyzed by S-SMase at pH 7.4 10-fold more than same donor plasma LDL, suggesting that LDL is modified in the arterial wall to increase its susceptibility to S-SMase. In summary, atherogenic lipoproteins are excellent substrates for S-SMase, even at neutral pH, making this enzyme a leading candidate for the arterial wall SMase that hydrolyzes LDL-SM and causes subendothelial LDL aggregation.     

Acidic sphingomyelinase mediates entry of N. gonorrhoeae into nonphagocytic cells

Invasion of human mucosal cells by N. gonorrhoeae via the binding to heparansulfate proteoglycan receptors is considered a crucial event of the infection. Using different human epithelial cells and primary fibroblasts, we show here an activation of the phosphatidylcholine-specific phospholipase C (PC-PLC) and acidic sphingomyelinase (ASM) by N. gonorrhoeae, resulting in the release of diacylglycerol and ceramide. Genetic and/or pharmacological blockade of ASM and PC-PLC cause inhibition of cellular invasion by N. gonorrhoeae. Complementation of ASM-deficient fibroblasts from Niemann-Pick disease patients restored N. gonorrhoeae-induced signaling and entry processes. The activation of PC-PLC and ASM, therefore, is an essential requirement for the entry of N. gonorrhoeae into distinct nonphagocytic human cell types including several epithelial cells and primary fibroblasts.     

The distal pathway of lipoprotein-induced cholesterol esterification, but not sphingomyelinase-induced cholesterol esterification, is energy-dependent

The stimulation of the intracellular cholesterol esterification pathway by atherogenic lipoproteins in macrophages is a key step in the development of atheroma foam cells. The esterification pathway can also be stimulated by hydrolysis of cell-surface sphingomyelin by the enzyme sphingomyelinase (SMase). In both cases, intracellular cholesterol transport to the cholesterol esterifying enzyme, acyl-CoA:cholesterol O-acyltransferase (ACAT), is thought to be critical, although the mechanism of cholesterol transport is not known. In this report, we explore two fundamental properties of the cholesterol esterification pathway, namely its dependence on energy and the effect of other treatments that block membrane vesicle trafficking. After the atherogenic lipoprotein, beta-very low density lipoprotein (beta-VLDL), was internalized by macrophages and hydrolyzed in lysosomes, the cells were depleted of energy by treatment with sodium azide and 2-deoxyglucose or by permeabilization. Under these conditions, which allowed equal beta-VLDL-cholesteryl ester hydrolysis, cholesterol esterification was markedly decreased in the energy-depleted cells. This effect was not due to blockage of lysosomal cholesterol export. In the permeabilized cell system, energy repletion restored beta-VLDL-induced cholesterol esterification. Remarkably, stimulation of cholesterol esterification by SMase was not inhibited by energy depletion. Energy depletion also inhibited beta-VLDL-induced, but not SMase-induced, cholesterol esterification in Chinese hamster ovary cells. Similar experiments were carried out using N-ethylmaleimide, low potassium medium, or inhibitors of phosphatidylinositol 3-kinase, each of which blocks intracellular membrane vesicle trafficking. These treatments also inhibited beta-VLDL-induced, but not SMase-induced, cholesterol esterification. Finally, we show here that SMase treatment of cells leads to an increase in plasma membrane vesiculation that is relatively resistant to energy depletion. In summary, the stimulation of cholesterol esterification by lipoproteins, but not by SMase, is energy-dependent, N-ethylmaleimide-sensitive, and blocked by both low potassium and phosphatidylinositol 3-kinase inhibitors. The affected step or steps are distal to cholesterol export from lysosomes and not due to direct inhibition of the ACAT enzyme. Thus, the mechanisms involved in lipoprotein-induced versus SMase-induced cholesterol esterification are different, perhaps due to the involvement of energy-dependent vesicular cholesterol transport in the lipoprotein pathway and a novel, energy-independent vesicular transport mechanism in the SMase pathway.     

Degranulation plays an essential part in regulating cell surface expression of Fas ligand in T cells and natural killer cells

Fas ligand (FasL) triggers apoptosis during cytotoxicity mediated by cytotoxic T lymphocytes and during immune downregulation. The ability of T cells and natural killer cells to trigger apoptosis through this mechanism is controlled by the cell surface expression of FasL (ref. 2). Because FasL expression is up-regulated on activation, FasL was thought to be delivered directly to the cell surface. Here we show that newly synthesized FasL is stored in specialized secretory lysosomes in both CD4+ and CD8+ T cells and natural killer cells, and that polarized degranulation controls the delivery of FasL to the cell surface. In this way, FasL-mediated apoptosis is finely controlled by receptor-mediated target-cell recognition. The cytoplasmic tail of FasL contains signals that sort FasL to secretory lysosomes in hemopoietic cells. This pathway may provide a general mechanism for controlling the cell surface appearance of proteins involved in immune regulation.     

Glutathione regulation of neutral sphingomyelinase in tumor necrosis factor-alpha-induced cell death

Tumor necrosis factor-alpha (TNFalpha)-induced cell death involves a diverse array of mediators and regulators including proteases, reactive oxygen species, the sphingolipid ceramide, and Bcl-2. It is not known, however, if and how these components are connected. We have previously reported that GSH inhibits, in vitro, the neutral magnesium-dependent sphingomyelinase (N-SMase) from Molt-4 leukemia cells. In this study, GSH was found to reversibly inhibit the N-SMase from human mammary carcinoma MCF7 cells. Treatment of MCF7 cells with TNFalpha induced a marked decrease in the level of cellular GSH, which was accompanied by hydrolysis of sphingomyelin and generation of ceramide. Pretreatment of cells with GSH, GSH-methylester, or N-acetylcysteine, a precursor of GSH biosynthesis, inhibited the TNFalpha-induced sphingomyelin hydrolysis and ceramide generation as well as cell death. Furthermore, no significant changes in GSH levels were observed in MCF7 cells treated with either bacterial SMase or ceramide, and GSH did not protect cells from death induced by ceramide. Taken together, these results show that GSH depletion occurs upstream of activation of N-SMase in the TNFalpha signaling pathway. TNFalpha has been shown to activate at least two groups of caspases involved in the initiation and "execution" phases of apoptosis. Therefore, additional studies were conducted to determine the relationship of GSH and the death proteases. Evidence is provided to demonstrate that depletion of GSH is dependent on activity of interleukin-1beta-converting enzyme-like proteases but is upstream of the site of action of Bcl-2 and of the execution phase caspases. Taken together, these studies demonstrate a critical role for GSH in TNFalpha action and in connecting major components in the pathways leading to cell death.     

Mg2+ binding and catalytic function of sphingomyelinase from Bacillus cereus

The modes of Mg2+ binding to SMase from Bacillus cereus were studied on the basis of the changes in the tryptophyl fluorescence intensity. This enzyme was shown to possess at least two binding sites for Mg2+ with low and high affinities. The effects of Mg2+ binding on the enzymatic activity and structural stability of the enzyme molecule were also studied. The results indicated that the binding of Mg2+ to the low-affinity site was essential for the catalysis, but was independent of the substrate binding to the enzyme. It was also indicated that the alkaline denaturation of the enzyme was partly prevented by the Mg2+ binding, whereas no significant protective effect was observed against the denaturation by urea. The pH dependence of the kinetic parameters for the hydrolysis of micellar HNP and mixed micellar SM with Triton X-100 (1:10), catalyzed by SMase from B. cereus, was studied in the presence of a large amount of Mg2+ to saturate both the low- and high-affinity sites. The pH dependence curves of the logarithm of 1/Km for these two kinds of substrates were similar in shape to each other, and showed a single transition. On the other hand, the shapes of the pH dependence curves of the logarithm of kcat for these two kinds of substrates were different from each other. The pH dependence curve for micellar HNP showed three transitions and, counting from the acidic end of the pH region, the first and third transitions having tangent lines with slopes of +1 and -1, respectively. On the other hand, the curve for mixed micellar SM with Triton X-100 showed one large transition with a slope of +1 (the first transition) and a very small transition (the third transition). On the basis of the present results and the three-dimensional structure of bovine pancreatic DNase I, which has a primary structure similar to that of B. cereus SMase, we proposed a catalytic mechanism for B. cereus SMase based on general-base catalysis.     

TNF receptor death domain-associated proteins TRADD and FADD signal activation of acid sphingomyelinase

Sphingomyelinase (SMase) activation and ceramide generation have emerged as an important signaling pathway transducing diverse biological effects of cytokine receptors like p55 tumor necrosis factor (TNF) receptor or Fas. Here we describe the TNF-dependent activation of acid SMase (A-SMase) through the p55 TNF receptor-associated proteins TRADD and FADD. Overexpression of TRADD and FADD in 293 cells did not change basal activity of A-SMase but enhanced TNF-induced stimulation of A-SMase. Other TNF R55-associated proteins like TRAF2 and RIP, which were reported to mediate TNF R55-mediated activation of nuclear factor kappaB, did not affect activation of A-SMase. Caspase inhibitors markedly reduced A-SMase activity, suggesting the involvement of an ICE-like protease in TRADD/FADD-mediated activation of A-SMase. Overexpression of caspase-8/a (FLICE/MACH) or caspase-10/b (FLICE2) did not change A-SMase activity, suggesting that TRADD/FADD-mediated activation of A-SMase involves a yet to be defined caspase-like protease distinct from caspase-8/a or -10/b.     

Functional characterization of the N-glycosylation sites of human acid sphingomyelinase by site-directed mutagenesis

Most soluble lysosomal enzymes require a mannose-6-phosphate recognition marker present on asparagine-linked oligosaccharides for proper targeting to lysosomes. We have determined the influence of the six potential N-linked oligosaccharide chains of human acid sphingomyelinase (ASM) on catalytic activity, targeting, and processing of the enzyme. Each N-glycosylation site was modified by site-directed mutagenesis and subsequently expressed in COS-1 cells. Evidence is presented that five of these sites are used. Elimination of the four N-terminal glycosylation sites does not disturb lysosomal targeting, processing, or enzymatic activity. However, removal of the two C-terminal N-glycosylation sites inhibits the formation of mature enzyme. Absence of glycosylation site five resulted in rapid cleavage of the primary translation product to an enzymatically inactive protein which accumulated inside the endoplasmic reticulum/Golgi, whereas deletion of glycosylation site six led to the formation of an inactive ASM precursor, also retained inside the endoplasmic reticulum/Golgi. Our results also provide evidence that the site of early proteolytic cleavage of newly synthesized ASM must be located between the second and third glycosylation sites.     

Negative regulation of interleukin-1beta-activated neutral sphingomyelinase by protein kinase C in rat mesangial cells

Endogenous ceramide is produced by the action of acidic or neutral sphingomyelinases (SMase) in response to stimuli such as proinflammatory cytokines or other inducers of stress. Interleukin-1beta (IL-1beta) is known to stimulate ceramide formation in rat renal mesangial cells; however, the respective subtype of SMase and its regulation have not been investigated. We found that IL-1beta induced an increase in endogenous ceramide levels via the action of a neutral SMase but not an acidic SMase in rat mesangial cells. Cytokine-induced activation of neutral SMase was inhibited by stimulation of protein kinase C (PKC) by the phorbol ester TPA which caused a reduction of ceramide back to control levels. This inhibitory effect of TPA was reversed by the specific PKC-inhibitor Ro-318220. Long-term incubation (24 h) of mesangial cells with TPA, which downregulates PKC-alpha, -delta, and -epsilon isoenzymes, resulted in a recovery of IL-1beta-stimulated neutral SMase activity as well as ceramide formation. These data implicate an important modulatory function of PKC in ceramide production in IL-1beta-activated mesangial cells.     

Septal innervation of mossy cells in the hilus of the rat dentate gyrus: an anterograde tracing and intracellular labeling study

To define the cellular processing of human cystatin C as well as to lay the groundwork for investigating its contribution to lcelandic Hereditary Cerebral Hemorrhage with Amyloidosis (HCHWA-I), we have characterized the trafficking, secretion, and extracellular fate of human cystatin C in transfected Chinese hamster ovary (CHO) cells. It is constitutively secreted with an intracellular half-life of 72 min. Gel filtration of cell lysates revealed the presence of three cystatin C immunoreactive species; an 11 kDa species corresponding to monomeric cystatin C, a 33 kDa complex that is most likely dimeric cystatin C and immunoreactive material, > or = 70 kDa, whose composition is unknown. Intracellular monomeric cystatin C is functionally active as a cysteine protease inhibitor, while the dimer is not. Medium from the transfected CHO cells contained only active monomeric cystatin C indicating that the cystatin C dimer, formed during intracellular trafficking, is converted to monomer at or before secretion. Cells in which exit from the endoplasmic reticulum (ER) was blocked with brefeldin A contained the 33 kDa species, indicating that cystatin C dimerization occurs in the ER. After removal of brefeldin A, there was a large increase in intracellular monomer suggesting that dimer dissociation occurs later in the secretion pathway, after exiting the ER but prior to release from the cell. Extracellular monomeric cystatin C was found to be internalized into lysosomes where it again dimerized, presumably as a consequence of the low pH of late endosome/lysosomes. As a dimer, cystatin C would be prevented from inhibiting the lysosomal cysteine proteases. These results reveal a novel mechanism, transient dimerization, by which cystatin C is inactivated during the early part of its trafficking through the secretory pathway and then reactivated prior to secretion. Similarly, its uptake by the cell also leads to its redimerization in the lysosomal pathway.     

Distinct adapter proteins mediate acid versus neutral sphingomyelinase activation through the p55 receptor for tumor necrosis factor

Ceramide, generated by the enzymatic function of sphingomyelinases (SMases) has emerged as an important signaling pathway transducing diverse biological effects of various cytokine receptors. The 55-kDa receptor for tumor necrosis factor (TNF-R55) activates two types of SMases through distinct cytoplasmic domains. The death domain that is responsible for the initiation of the apoptotic pathway also signals for the activation of an acid SMase (A-SMase). The adapter protein TRADD binds to TNF-R55 in a ligand-dependent manner and serves as anchor for the subsequent recruitment of other proteins into the signaling complex that directly lead to cell death or nuclear factor-kappaB (NF-kappaB) induction. Notably, the two pro-apoptotic adapter proteins TRADD and FADD are also involved in the activation of A-SMase. In contrast, the NF-kappaB-inducing adapters TRAF2 and RIP do not signal for A-SMase. Thus, activation of A-SMase appears to belong to signals leading to TNF-induced cell death. A second signaling domain (NSD) is located upstream of the death domain and directly links the TNF-R55 to the activation of a neutral SMase (N-SMase). A novel adapter protein, FAN, has been identified that specifically binds to the NSD. FAN contains five WD repeats at its carboxy terminus, while it shows significant sequence homology with the mouse beige protein and its human homolog, the CHS protein, in the center portion of the protein. Overexpression of full-length FAN enhanced N-SMase activity in TNF-treated cells, whereas truncated mutants of FAN produced dominant negative effects. FAN, however, did not interfere with any of the TNF responses signaled for by the death domain. Taken together, our data suggest that distinct cytoplasmic domains of TNF-R55 initiate independent signaling pathways by binding different adapter proteins.     

Acid sphingomyelinase is not essential for the IL-1 and tumor necrosis factor receptor signaling pathway leading to NFkB activation

A recent report has suggested that tumor necrosis factor (TNF) utilizes acid sphingomyelinase (SMase) pathway to activate NFkB (Schutze et al. 1992. Cell 71:765). To directly investigate the role of acid SMase in IL-1 and TNF receptor-mediated signal transduction, we examined the ability of Niemann-Pick disease (NPD) type A fibroblasts, which are deficient in acid SMase, to induce IL-8 gene expression through activating NFkB. Unexpectedly, IL-1 alpha and TNF-alpha efficiently induced IL-8 production and IL-8 mRNA in NPD type A fibroblasts as in normal fibroblasts. Furthermore, activation of NFkB was also induced in NPD type A fibroblasts in response to IL-1 alpha and TNF-alpha stimulation to a similar extent as in normal fibroblasts. These results provide evidence that acid SMase is not essential in IL-1 and TNF receptor signaling leading to NFkB activation as well as the cytokine gene activation which is regulated by NFkB.     

A distant evolutionary relationship between bacterial sphingomyelinase and mammalian DNase I

The three-dimensional structure of bacterial sphingomyelinase (SMase) was predicted using a protein fold recognition method; the search of a library of known structures showed that the SMase sequence is highly compatible with the mammalian DNase I structure, which suggested that SMase adopts a structure similar to that of DNase I. The amino acid sequence alignment based on the prediction revealed that, despite the lack of overall sequence similarity (less than 10% identity), those residues of DNase I that are involved in the hydrolysis of the phosphodiester bond, including two histidine residues (His 134 and His 252) of the active center, are conserved in SMase. In addition, a conserved pentapeptide sequence motif was found, which includes two catalytically critical residues, Asp 251 and His 252. A sequence database search showed that the motif is highly specific to mammalian DNase I and bacterial SMase. The functional roles of SMase residues identified by the sequence comparison were consistent with the results from mutant studies. Two Bacillus cereus SMase mutants (H134A and H252A) were constructed by site-directed mutagenesis. They completely abolished their catalytic activity. A model for the SMase-sphingomyelin complex structure was built to investigate how the SMase specifically recognizes its substrate. The model suggested that a set of residues conserved among bacterial SMases, including Trp 28 and Phe 55, might be important in the substrate recognition. The predicted structural similarity and the conservation of the functionally important residues strongly suggest a distant evolutionary relationship between bacterial SMase and mammalian DNase I. These two phosphodiesterases must have acquired the specificity for different substrates in the course of evolution.     

Effects of Ara-C on neutral sphingomyelinase and mitogen- and stress-activated protein kinases in T-lymphocyte cell lines

Neutral sphingomyelinase (SMase) can be activated by extracellular signals to produce ceramide, which may affect mitogen-activated protein kinase (MAPK) activities. Neutral SMase activity was assessed in membranes from Jurkat, a human T-cell line, and EL4, a murine T-cell line. Ara-C activated SMase with 10 minutes in both Jurkat and EL4 cells, while phorbol ester (PMA) had no effect. PMA, but not Ara-C or ceramides, activated ERK MAPKS, in Jurkat and EL4. PMA acted synergistically with ionomycin to activate JNK MAPKs in Jurkat and EL4 within 10 minutes. Ara-C activated JNKs only after prolonged incubation (90-120 minutes). Thus, ceramide is not a positive signal for ERK activation in T-cell lines. The effects of Ara-C on JNK activity may be mediated through secondary response pathways.     

Lysosomal enzyme trafficking between phagosomes, endosomes, and lysosomes in J774 macrophages. Enrichment of cathepsin H in early endosomes

In this study we take advantage of recently developed methods using J774 macrophages to prepare enriched fractions of early endosomes, late endosomes, dense lysosomes, as well as phagosomes of different ages enclosing 1-micron latex beads to investigate the steady state distribution and trafficking of lysosomal enzyme activity between these organelles. At steady state these cells appear to possess four different cellular structures, in addition to phagolysosomes, where acid hydrolases were concentrated. The first site of hydrolase concentration was the early endosomes, which contained the bulk of the cellular cathepsin H. This enzyme was acquired by phagosomes significantly faster than the other hydrolases tested. The second distinct site of lysosomal enzyme concentration was the late endosomes which contain the bulk of cathepsin S. The third and fourth large pools of hydrolases were found in two functionally distinct types of dense lysosomes, only one of which was found to be secreted in the presence of chloroquine or bafilomycin. Among this secreted pool was soluble furin, generally considered only as a membrane-bound trans-Golgi network resident protein. Thus, the organelles usually referred to as "lysosomes" in fact encompass a growing family of highly dynamic but functionally distinct endocytic organelles.     

Purification and characterization of a membrane bound neutral pH optimum magnesium-dependent and phosphatidylserine-stimulated sphingomyelinase from rat brain

Sphingomyelin hydrolysis and ceramide generation catalyzed by sphingomyelinases (SMase) are key components of the signaling pathways in cytokine- and stress-induced cellular responses. In this study, we report the partial purification and characterization of the membrane bound, neutral pH optimal, and magnesium-dependent SMase (N-SMase) from rat brain. Proteins from Triton X-100 extract of brain membrane were purified sequentially with DEAE-Sephacel, heparin-Sepharose, ceramic hydroxyapatite, Mono Q, phenyl-Superose, and Superose 12 column chromatography. After eight purification steps, the specific activity of the enzyme increased by 3030-fold over the brain homogenate. The enzyme hydrolyzed sphingomyelin but not phosphatidylcholine and its activity was dependent upon magnesium with an optimal pH of 7.5 and a native pI of 5.2. Delipidation of the enzyme through chromatographic purification or by extraction with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid followed by gel filtration revealed that the enzyme became increasingly dependent on phosphatidylserine (PS). Up to 20-fold stimulation was observed with PS whereas other lipids examined were either ineffective or only mildly stimulatory. The Km of the enzyme for substrate sphingomyelin (3.4 mol %) was not affected by PS. The highly purified enzyme was inhibited by glutathione with a >95% inhibition observed with 3 mM glutathione and with a Hill number calculated at approximately 8. The significance of these results to the regulation of N-SMase is discussed.     

Isoproterenol evokes extracellular Ca2+ spikes due to secretory events in salivary gland cells

Secretory cells should in principle export substantial amounts of calcium via exocytosis since Ca2+ is sequestered in secretory granules. Based on a new technique for measurements of the extracellular calcium concentration in the vicinity of the cell membrane and on the droplet technique, we have monitored the rate of calcium extrusion from salivary gland acinar cells. Isoproterenol (ISP), a beta-adrenergic agonist and powerful secretogogue, evoked no change in the cytosolic free Ca2+ concentration ([Ca2+]i) but induced vigorous extracellular Ca2+ concentration ([Ca2+]i) spiking. The absence of [Ca2+]i elevation and the pulsatile nature of the changes in [Ca2+]i indicate that these spikes are most likely due to calcium release from secretory granules. The cholinergic agonist acetylcholine (ACh), which induces moderate secretion, evoked a marked rise in [Ca2+]i and a smooth rise in [Ca2+]i, most likely induced by plasma membrane calcium pumps, on which shortlasting [Ca2+]i spikes were superimposed. The rate of ISP-induced calcium efflux was very substantial. The calculated calcium loss during the first 100 s of supramaximal stimulation corresponded to a reduction of the total cellular calcium concentration of approximately 0.4 mM. We conclude that in salivary glands, calcium release via exocytosis is one of the main mechanisms extruding calcium from cells to the extracellular milieu.     

Purification of a newly identified alkaline sphingomyelinase in human bile and effects of bile salts and phosphatidylcholine on enzyme activity

The hydrolysis of sphingomyelin (SM) generates important signals regulating cell proliferation and apoptosis. Acid and neutral sphingomyelinases (SMase) have been identified and their biological effects intensively studied. We recently found in human bile a novel alkaline SMase that may have important roles in hepatobiliary diseases. In this work, we purified the enzyme and studied the factors influencing enzyme activity. Purification steps included Sephadex G25, diethylaminoethyl (DEAE)-Sepharose, Sephacryl S-200, and sphingosylphosphorylcholine (SPC) affinity chromatographies. A single protein of 92 kd was obtained with the specific enzyme activity increased 1,154-fold. The enzyme specifically hydrolyzed SM to ceramide, had a weaker activity against phosphatidylcholine (PC), and no activity against either phosphatidylethanolamine (PE) or p-nitrophenyl phosphate. Its optimum pH was 9.0 and its Vmax and Km were 45 micromol/h/mg and 2.5 x 10(-5) mol/L, respectively. The enzyme activity was dependent on concentration and structure of bile salts. Both trihydroxy and dihydroxy bile salts at concentrations up to their critical micellar concentrations activated the alkaline SMase, trihydroxy bile salts being more potent than dihydroxy ones. The side chain of trihydroxy bile salts was also important. Taurocholate (TC) was most effective in activating SMase, followed by glycocholate (GC), and cholate. 3-((3-cholamidopropyl)dimethylammonio)-propanesulfonate (CHAPS) alone had no effect on SMase activity but inhibited TC-induced activation of SMase. PC competitively inhibited bile alkaline SMase activity, with the 50% inhibition occurring at a PC/SM ratio of approximately 28. In conclusion, we purified a novel alkaline SMase from human bile and found that its activity is dependent on the levels of two major biliary components: PC and bile salts.