Sphingolipids have recently been shown to be important participants in the regulation of a variety of cellular processes (1, 2, 3) . Sphingosine, one of the metabolites of sphingolipids, was in its early studies demonstrated as a potent endogenous inhibitor of protein kinase C (1, 4) and has been implicated to be a negative regulator for a few signaling processes(1, 4) . Further studies, however, revealed that the exogenous sphingosine also induces various types of positive biological actions, e.g. activation of phospholipase D(5) , stimulation of cell proliferation(6) , regulation of Ca mobilization from the internal pool (7, 8, 9, 10, 11) , and inhibition of Ca influx through the plasma membranes(12) . These actions seem to be exerted through phosphatidate (5) or a phosphorylated product of sphingosine, sphingosine 1-phosphate (S1P) ()(7, 8, 13, 14, 15) ; many of them were suggested to be independent of protein kinase C. S1P was reported to act directly on the internal Ca pool resulting in Ca mobilization in a way similar to inositol 1,4,5-trisphosphate(8, 15) . This lysosphingolipid has also been proposed as a second messenger of platelet-derived growth factor and serum on cell proliferation in fibroblasts(16) . In the brain and other peripheral tissues of inherited sphingolipid disorders, it has been shown that any one of lysosphingolipids, e.g. sphingosylphosphorylcholine (SPC), galactosylsphingosine (psychosine), or glucosylsphingosine (glucopsychosine), is accumulated(4, 17, 18, 19) . These lysosphingolipids might be responsible for the respective pathogenesis(4, 17, 18, 19) . SPC has recently been shown, similarly to S1P, to be a potent Ca releaser from the internal pool and suggested to cause the Ca release from the 1,4,5-trisphosphate-sensitive pool in various cell types(7, 8, 9, 10) .

These observations suggest that, in addition to protein kinase C inhibition, intracellular Ca mobilization is an important action of lysosphingolipids, which may have pathological and physiological significance. This raises the question of whether the Ca mobilization is caused by the activation of the phospholipase C-Ca signal transduction pathway. In fact, recent studies demonstrated that extracellular S1P in Swiss 3T3 fibroblasts (15) and sphingosine in Swiss 3T3 fibroblasts (5) , astrocytes(20) , and foreskin fibroblasts (21) can induce inositol phosphate production, probably reflecting activation of phospholipase C. Although the S1P-induced [Ca]increase in the cells has been suggested to occur independently of the enzyme activation(15) , at least a part of the sphingosine-induced Ca mobilization as well as phospholipase C activation in foreskin fibroblasts was sensitive to PTX, showing some similarity to a typical feature of PTX-sensitive G-protein-mediated activation of the phospholipase C-Ca pathway (21) . If this is the case, we might be allowed to imagine the presence of a receptor(s) for the lysosphingolipids which lead to the activation of phospholipase C, although the previous findings have not excluded the possibility that the lipids penetrate into the cells and act on the pathway inside the cells.

In the present paper, our study was focussed on the Ca mobilizing actions of SPC and other lysosphingolipids which are accumulated in the respective sphingolipidosis, especially on the mechanisms of their actions. We found that, in HL60 leukemia cells, extracellularly added lysosphingolipids at 30 µM or less induced a rapid and transient increase in [Ca], the features of which are indistinguishable from those of the Ca response induced by UTP, a P-purinergic agonist, in the same cells. The transient [Ca] rises were associated with inositol phosphate production, and both Ca and inositol phosphate responses were inhibited by the treatments of cells with PTX and U73122, a potent phospholipase C inhibitor. Our results suggest that extracellular lysosphingolipids at appropriate doses induce a [Ca] rise due to the activation of the phospholipase C being mediated by a putative receptor(s) coupled to a PTX-sensitive G-protein(s).

EXPERIMENTAL PROCEDURES

Materials

Purity Check and Purification of SPC and Psychosine

Figure 2: Effects of sphingomyelinase treatment and TLC purification of SPC and psychosine on Ca mobilization. In A, 3 mM SPC and psychosine were incubated at 37 °C for 1 h with or without sphingomyelinase (SMase, 0.2 units) in 10 mM Tris-HCl buffer (pH 7.4), containing 2 mM MgCl, 10% MeOH, 0.8% glycerol in a final volume of 100 µl. A part (2 µl) of the reaction mixture was analyzed by Silica Gel 60 TLC in solvent I as described under ``Experimental Procedures.'' Lane 1, SPC (without SMase); lane 2, SPC (with SMase); lane 3, SPC (with SMase) plus authentic sphingosine; lane 4, authentic sphingosine; lane 5, TLC-purified SPC; lane 6, psychosine (without SMase); lane 7, psychosine (with SMase). In B, unpurified psychosine (lane 1) and TLC-purified psychosine (lane 2) were analyzed by Silica Gel 60 TLC in solvent I as described under ``Experimental Procedures.'' In A and B, the position of origin (O), front (F), SPC, psychosine (PSY), or sphingosine was marked as an arrow. In C and D, [Ca] response to SPC or psychosine (PSY) treated with or without SMase, TLC-purified SPC, or TLC-purified psychosine was measured as indicated. Concentration would be 30 µM unless degradation occurred during enzyme treatment.

Cell Cultures

Figure 7: Differentiation into neutrophil-like cells attenuates phospholipase C and the subsequent Ca mobilization in response to SPC and psychosine. In A, cell membranes were prepared from undifferentiated cells (a) and neutrophil-like cells differentiated by dibutyryl cyclic AMP (b). Their cholate extracts were subjected to a SDS-polyacrylamide gel electrophoresis, transferred to an Immobilon sheet, and then probed with G- or G-specific antiserum as described under ``Experimental Procedures.'' In B and C, representative traces of [Ca]changes in undifferentiated cells (B) and neutrophil-like cells (C) (non-treated (a and b) or treated (c) with PTX) are shown. At arrows, 10 nM formyl-Met-Leu-Phe (FMLP) or 30 µM SPC was added to the incubation medium, as indicated. In D, [Ca]changes caused for 2 min by 30 µM SPC and psychosine, and for 10 min by AlF (10 mM NaF plus 10 µM AlCl) in differentiated cells (neutrophil-like cells) were compared with those in undifferentiated cells. The results are expressed as percentages of those in undifferentiated cells. In an inset, a typical trace of [Ca] change by AlF in undifferentiated (a) and neutrophil-like (b) cells is shown. In E, undifferentiated cells (open column or ) and neutrophil-like cells (closed column or ) both labeled with [H]inositol were incubated for 1 min without or with formyl-Met- Leu-Phe (10 nM), SPC (30 µM), or psychosine (30 µM). Production of IP + IP was measured. Results are expressed as percentages of the respective basal value obtained without any addition. Normalized basal values (cpm) were 580 ± 35 and 472 ± 73 for undifferentiated cells and the neutrophil-like cells, respectively. In the inset, time courses of AlF (10 mM NaF plus 10 µM AlCl)-induced response (IP + IP) are shown. Results are expressed as percentages of the respective initial value. Data are means ± S.E. of three separate experiments.

Measurement of [H]Inositol Phosphates Production

Measurement of [Ca]

Immunoblot Analysis

Data Presentation

RESULTS

Extracellular SPC and Psychosine Increase [Ca]i in a Manner Sensitive to PTX

Figure 1: Effect of PTX on SPC and psychosine-induced increase in [Ca]. A, control cells, non-treated with PTX, and B, PTX-treated cells, show traces of time-dependent [Ca] changes each representing the changes induced by UTP (1 µM) or the indicated doses (µM) of SPC, psychosine (PSY) or sphingosine. C-E show dose-dependent increase in [Ca] (=peak value - basal value) with SPC (C), PSY (D), and sphingosine (E) in control cells () and PTX-treated cells (). Results are means ± S.E. of five separate experiments. F shows [Ca] obtained by 1 µM UTP in a control (open column) and PTX-treated (closed column) cells. Results are means ± S.E. of seven separate experiments.

As mentioned in the Introduction, another well documented action of lysosphingolipids is protein kinase C inhibition, especially in the earlier period of the studies(4) . SPC and psychosine therefore might induce Ca mobilization as a result of the enzyme inhibition. To examine this possibility, we also used sphingosine which is a similar or more potent inhibitor of protein kinase C than SPC or psychosine(4) . Sphingosine also increased [Ca]; however, the time course was very slow and the net increase was much less than that induced by similar doses of SPC and psychosine (Fig. 1A). Moreover, the sphingosine-induced [Ca] increase was totally insensitive to PTX (Fig. 1, B and E). We also examined the effect of another protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), but this drug never increased [Ca] (data not shown). In fibroblasts(11, 21) , the sphingosine-induced [Ca] rise has been suggested to be protein kinase C-independent. These results suggest that at least the PTX-sensitive increase in [Ca] by SPC and psychosine is independent of the protein kinase C inhibition.

TLC analysis of the SPC sample showed the presence of a small, but detectable, amount of unknown compound that is positive with ninhydrin at R = 0.26 (Fig. 2A, lane 1). However, it was confirmed that SPC itself elicited the Ca response and the contaminated unknown compound is inactive to induce the response (Fig. 2). Sphingomyelinase from B. cereus almost completely converted SPC to sphingosine, but did not influence the unknown compound (Fig. 2A, lanes 2-4). The enzyme-treated SPC never elicited a rapid and transient [Ca] increase which is a characteristic to the untreated SPC, instead induced a rather slow increase probably due to sphingosine (Fig. 2C). The enzyme was rather specific to SPC; psychosine was tolerable to the enzyme (Fig. 2A, lanes 6 and 7) and the lipid-induced [Ca] increase was unaffected by its treatment (Fig. 2C). Furthermore, the TLC-purified SPC sample, which is free from the unknown compound (Fig. 2A, lane 5), induced the Ca response to an extent similar to that of the unpurified SPC (Fig. 2C).

Although psychosine obtained from the drug company showed a single spot that is positive with ninhydrin and anthrone/HSO on TLC using two solvent systems, we further purified the psychosine by TLC (Fig. 2B, lanes 1 and 2). The purified psychosine also induced a rapid and transient [Ca] increase as effectively as the unpurified psychosine did (Fig. 2D). Since the active compound to induce Ca mobilization was demonstrated to be SPC or psychosine itself and furthermore, there was no appreciable difference in the ability to induce Ca response between purified and unpurified products, we performed the following experiments without further purification of the lipids.

SPC and Psychosine Mobilize Cafrom the Internal Pool in a Manner Sensitive to U73122, a Phospholipase C Inhibitor

Figure 3: Effects of extracellular Ca and U73122 on SPC, psychosine, and UTP-induced increase in [Ca]. Representative traces of [Ca] change from three or four separate experiments by 30 µM SPC (A), 30 µM psychosine (B), and 1 µM UTP (C) in the absence or presence of 2.5 mM EGTA (as shown with ``E'' in the panel) or 2.5 µM U73122 (as shown with ``U73'' in the panel) are shown.

SPC and Psychosine Produce Inositol Phosphate

Figure 4: Time-dependent effect of SPC, psychosine, and UTP on inositol phosphate production. The cells labeled with [H]inositol were incubated for the indicated times without () or with 30 µM SPC (), 30 µM psychosine (), or 1 µM UTP () in the cells non-treated (A, C, and E) or treated (B, D, and F) with PTX. Results are expressed as percentages of the respective initial value. Normalized initial values (cpm) in control cells were 465 ± 16, 206 ± 6, and 473 ± 16 for IP, IP, and IP, respectively. These values were not significantly changed by PTX treatment. All data are means ± S.E. of three separate experiments.

Figure 5: Dose-response curves of SPC and psychosine on inositol phosphate production and its suppression by PTX and U73122. The cells labeled with [H]inositol were incubated for 1 min with the indicated doses of SPC (A) and psychosine (PSY) (B) in the cells non-treated (, ) and treated () with PTX. In some experiments, U73122 () (2.5 µM) was added to the incubation medium 2 min before SPC and PSY addition. Production of IP plus IP was measured. Results are expressed as percentages of the basal values obtained without test agents. Normalized basal values (cpm) were 554 ± 16 and 552 ± 16 for the cells non-treated and treated with PTX, respectively. Data are means ± S.E. of three separate experiments.

PTX and U73122-sensitive Activation of Phospholipase C Is not Secondary to the [Ca]i Rise

Figure 6: Thapsigargin and ionomycin induced [Ca] increase in a manner independent of phospholipase C and insensitive to PTX. [Ca] change in the cells non-treated (A) and treated (B) with PTX was monitored. At arrows, 300 nM thapsigargin as shown with ``TG,'' 1 µM ionomycin or 2.5 µM U73122 as shown with ``U73'' were added. The results shown are representative of three separate experiments. In C, the cells labeled with [H]inositol were incubated for the indicated times without (), with 300 nM thapsigargin (), 1 µM ionomycin (), or 30 µM SPC (). Results (IP + IP production) are expressed as percentages of initial values. Data are means ± S.E. of three separate experiments.

Differentiation into Neutrophil-like Cells Was Associated with Attenuation of Responses to Lysosphingolipids

As shown in Fig. 7A, the contents of G and G were actually increased by a dibutyryl cyclic AMP treatment of the cells as evidenced from increases in immunodetectable G and G. The dibutyryl cyclic AMP-treated cells also showed a PTX-sensitive formyl-Met-Leu-Phe-induced [Ca] increase (Fig. 7C) and inositol phosphate production (Fig. 7E), currently recognized to be differentiation markers. AlF, a non-selective G-protein activator, induces phospholipase C activation and the subsequent Ca mobilization in many types of cells(34, 36) . These AlF actions shown in Fig. 7, D and E, are very slow, but significant, and are slightly stronger in the differentiated cells than in the undifferentiated ones, probably reflecting higher contents of G proteins in the neutrophil-like differentiated cells than in the undifferentiated cells (Fig. 7, D and E). Unexpectedly, however, the SPC-induced Ca mobilization was markedly attenuated in the neutrophil-like cells (Fig. 7, B and C). The Ca response to psychosine was also decreased (Fig. 7D). In parallel with the Ca response, the inositol phosphate response to SPC and psychosine was clearly attenuated by differentiation, suggesting that the lipids signaling of the PTX-sensitive G-protein-coupled phospholipase C-Ca pathway is blocked before a G-protein step in the neutrophil-like cells (Fig. 7E).

Glucopsychosine and Lysosulfatides Also Induce CaMobilization and Inositol Phosphate Production

()

Figure 8: Effect of glucopsychosine and lysosulfatides on [Ca] and inositol phosphate production. In A (control cells non-treated with PTX) and B (PTX-treated cells), representative traces of [Ca] changes by the indicated doses of glucopsychosine (GlcPSY) and lysosulfatides (LSF) are shown. Where indicated, U73122 (2.5 µM) was added 2 min before addition of the respective lysosphingolipid. In C (control cells) and D (PTX-treated cells), the cells labeled with [H]inositol were incubated for the indicated times without (), with 30 µM lysosulfatides (), or with 30 µM glucopsychosine (). Results are expressed as percentages of initial values. Data are means ± S.E. of three separate experiments.

Lysoglycerophospholipids and Platelet-activating factor Also Induce CaMobilization, but They Were Less Effective Than Lysosphingolipids

DISCUSSION

In the present paper we have shown that lysosphingolipids (SPC, psychosine, glucopsychosine, and lysosulfatides) at doses lower than 30 µM induce phospholipase C activation and the subsequent Ca mobilization in a manner sensitive to PTX and U73122, a phospholipase C inhibitor. This is, to our knowledge, the first indication that these lysosphingolipids activate the phospholipase C-Ca system possibly through receptors coupling to a PTX-sensitive G-protein(s). The putative receptors may be different from the previously identified platelet-activating factor receptor (37) and lysophosphatidic acid receptor(38) .

As far as extracellular SPC-induced intracellular Ca mobilization is concerned, a few studies on fibroblasts (39) and FRTL-5 thyroid cells (40) have been reported. However, no significant production of inositol phosphate was observed in these experiments(39, 40) , despite the fact that Ca mobilizing receptor agonists, such as bradykinin, induced not only Ca mobilization to an extent similar to that with SPC but also phospholipase C activation under the same conditions(39) . In addition, in other studies, SPC mobilized Ca from permeabilized cells(7, 8, 9, 10) and purified endoplasmic reticulum membrane vesicles(8) . On the basis of these previous results, the SPC actions have been currently considered to occur inside the cells by the incorporated SPC molecules, without activating phospholipase C. The present results, however, suggest that at least the early phase of the Ca mobilization induced by lower than 30 µM SPC or other lysosphingolipids in intact HL60 cells is mediated by the activation of the enzyme. This suggestion is based on the following findings. First, SPC and other lysosphingolipids at doses lower than 30 µM induced immediate activation of phospholipase C. Second, U73122, a potent phospholipase C inhibitor, suppressed at least the early phase of the lysosphingolipids-induced increase in [Ca]. Third, treatment of the cells with either PTX or dibutyryl cyclic AMP attenuated both the lysosphingolipid-induced phospholipase C activation and Ca mobilization. Finally, phospholipase C activation is not a secondary response to the increase in [Ca]; agents such as thapsigargin and ionomycin, which primarily increase [Ca], never activated the enzyme in HL60 cells under the present conditions (Fig. 6).

Several findings in the present study suggest that the lysosphingolipids signaling is performed through G-protein-coupled receptors. The pattern and kinetics of [Ca] increase and inositol phosphate production by the lysosphingolipids were very similar to those of the responses to a G-protein-coupled receptor agonist, UTP (a P-purinergic agonist) (Fig. 1, Fig. 3, and Fig. 4). Furthermore, as stronger evidence for the involvement of G-protein coupled receptors, the lysosphingolipid actions are suppressed by prior treatment of the cells with PTX which, as is well known, ADP-ribosylates G-proteins and thereby blocks communication between receptors and effector enzymes. Similar PTX sensitivity has already been shown in the phospholipase C activation induced by several receptor agonists such as formyl-Met-Leu-Phe and UTP in leukocytes such as HL60 cells and neutrophils. This finding has been concluded to reflect the fact that receptors coupling to PTX-sensitive G-proteins mediate the phospholipase C activation(24, 25, 26, 32) . In this analogy, it is reasonable to assume that the lysosphingolipid actions are mediated via G-protein-coupled receptors. It is still possible, however, that amphipathic lysosphingolipids penetrate into the cells and then directly activate G-proteins. If this was the case, PTX would block the lipid-induced actions. This possibility is excluded from the experiments shown in Fig. 7. Dibutyryl cyclic AMP-induced differentiation into neutrophil-like cells enhanced AlF (a nonspecific G-protein activator)-induced phospholipase C activation, probably reflecting the increase in the amount of G-proteins. This suggests that in the differentiated cells, the downstream region of the G-protein-mediated signaling cascade leading to phospholipase C activation and Ca mobilization is rather fortified by the increase in PTX-sensitive G-proteins. On the contrary, the SPC and psychosine-induced enzyme activation was seriously suppressed by differentiation of the cells. This suggests that differentiation impairs the process between the action sites of lipids (or receptors) and G-proteins and hence may rule out the possibility that these lysosphingolipids directly activate G-proteins. Thus, the present pharmacological study suggests the existence of G-protein-coupled receptors for lysosphingolipids, although conclusive evidence for the existence of the receptors will have to await their molecular cloning.

In addition to lysosphingolipids, platelet-activating factor and some lysoglycerophospholipids, such as lysophosphatidylcholine and lysophosphatidic acid, also induced Ca mobilization in HL60 cells, but they were not as effective as SPC and other lysosphingolipids (Table 1). Furthermore, in contrast to SPC and psychosine effects which were attenuated in dibutyryl cAMP-induced differentiated cells (Fig. 7), platelet-activating factor-induced response was conversely enhanced by the induction of differentiation, suggesting that the putative receptors for lysosphingolipids are different from platelet-activating factor receptor. Among lysoglycerophospholipids examined, lysophosphatidylcholine was the most effective in the induction of Ca mobilization (Table 1). Similarly to the actions of lysosphingolipids, the lysophosphatidylcholine effect was PTX-sensitive, whereas the lysophosphatidic acid-induced response was not (Table 1). Thus, the receptor for lysophosphatidic acid (38) appears to be different from putative receptors for lysosphingolipids. On the other hand, it remains unclear whether lysoglycerophospholipids (including lysophosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidylinositol) other than lysophosphatidic acid share with lysosphingolipids the same receptor and signaling pathways.

Among lysosphingolipids, sphingosine and S1P have been previously shown to induce phospholipase C activation and the Ca mobilization in a few types of cells(5, 15, 20, 21) . In HL60 cells, sphingosine induced the Ca mobilization; however, this action was PTX-insensitive (Fig. 1). Furthermore, the [Ca] increase due to the lipid was so slow that it took 1-3 min to reach a peak value (Fig. 1). Thus, the sphingosine signaling pathway seems to be different from that of SPC and other lysosphingolipids. This also suggests that the PTX-sensitive Ca mobilization by lysosphingolipids cannot be explained by the inhibition of protein kinase C, because sphingosine is a protein kinase C inhibitor similar to or more potent than the lysosphingolipids examined in the present study(4) . We also preliminarily examined S1P actions on phospholipase C and the Ca mobilization in HL60 cells. This lipid also activated the enzyme and increased [Ca] in the cells. In this case, we could not detect any difference between S1P and SPC actions in their sensitivity to PTX and U73122. Thus, S1P seems to share a signaling pathway similar to that of SPC in HL60 cells. In Xenopus oocytes, however, S1P activated a Cl channel probably through phospholipase C activation, but SPC could not mimic the S1P action(41) . The receptor cloning again would make it clear whether all the lysosphingolipids and some lysoglycerophospholipids share the same receptor or each lipid interacts with its own receptor.

At the present stage of investigation, the physiological roles of the lysosphingolipid-induced activation of the phospholipase C-Ca pathway in leukocytes have not been clarified yet. This type of lysosphingolipid signaling was attenuated by dibutyryl cyclic AMP-induced differentiation of HL60 cells into neutrophil-like cells (Fig. 7). In the preliminary experiments, we found that other differentiation inducers such as dimethyl sulfoxide, retinoic acid, and vitamin D also diminished such lysosphingolipid signaling. This may suggest that only under undifferentiated conditions lysosphingolipids act as physiological and extracellular signals which are oriented to the phospholipase C-Ca pathway. In the previous study in differentiated cells such as fibroblasts (39, 42) and thyroid cells (40) , SPC has been shown to be a potent mitogen. The Ca mobilizing action of SPC may be involved in the cell proliferation(39, 40) . A preliminary finding in the undifferentiated HL60 cells, however, showed that SPC rather attenuated the cell growth and instead facilitated cell attachment to culture dishes. This phenomenon might reflect a physiological role of SPC as an inducer of cell differentiation. Further study is now in progress to clarify this point.

The possible existence of cell surface receptors for lysosphingolipids may allow consideration of a novel autocrine or paracrine regulatory mechanism operated by the lysosphingolipids in a way similar to other lipids mediators such as prostaglandins and leukotriens. At present, there are no data on the extracellular occurrence of lysosphingolipids in vivo. To establish the autocrine or paracrine role of the lipids, further studies on the problems are needed, which include characterization of intracellular and extracellular metabolic pathways and physiological functions of the lysosphingolipids as well as identification of their putative receptors.

FOOTNOTES

*

This work was supported in part by a research grant from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

()

The abbreviations used are: S1P, sphingosine 1-phosphate; PTX, pertussis toxin; [Ca], cytoplasmic free Ca concentration; SPC, sphingosylphosphorylcholine; G-protein, GTP-binding regulatory protein; IP, inositol monophosphate; IP, inositol bisphosphate; IP, inositol trisphosphate.

()

We should be cautious to interpret the fluorescence data in the case of the sustained pattern in our experimental conditions; it is possible that the amphipathic lipids induced the leakage of the fluorescence indicator. In fact, when we employed 300 µM SPC, the fluorescence intensity increased rapidly to the level near the maximal intensity even in the presence of 2.5 mM EGTA and its high level was sustained, where we found that the fluorescence indicator leaked into the medium. In contrast, in the case of the transient fluorescence change, the leakage of the indicator may not be always necessary to consider, because it is implausible that the leaky cells reuptake the leaked indicator. In addition, the early transient fluorescence changes by the lysosphingolipids shown in the present study were inhibitable by agents such as U73122 and PTX, rather specific agents for phospholipase C and G/G proteins, respectively.

ACKNOWLEDGEMENTS

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