Sphingosine and phorbol ester modulate protein kinase C activity and modify ATP-evoked calcium mobilization in glioma C6 cells.

The effect of sphingosine and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on ATP-evoked Ca(2+) mobilization in glioma C6 cells was studied with the Fura-2 video-imaging technique. Treatment of the cells with TPA, an activator of protein kinase C, reduced the ATP-evoked release of Ca(2+) from the intracellular stores, whereas sphingosine, known from in vitro studies as a protein kinase C inhibitor, potentiated Ca(2+) release synergistically with ATP. ATP-induced Ca(2+) mobilization was also enhanced by a specific protein kinase C inhibitor, GF 109203X. Pretreatment of the cells with GF 109203X prevented TPA action, whereas TPA diminished the stimulatory effect of sphingosine. However, this sphingosine effect was only observed after a short (1 min) treatment, whereas a longer treatment (5 min) reduced ATP-evoked Ca(2+) release. It is therefore concluded that sphingosine has two apparent actions: it inhibits protein kinase C providing a positive feedback regulation of receptor signals and it releases Ca(2+) from intracellular stores by an unknown mechanism, possibly independent of protein kinase C.     

Ethanol potentiates the stimulatory effects of phorbol ester, sphingosine and 4-hydroxynonenal on the hydrolysis of phosphatidylethanolamine in NIH 3T3 cells

Ethanol and other alcohols have been shown to specifically stimulate phospholipase-D-mediated hydrolysis of phosphatidylethanolamine (PtdEtn) in NIH 3T3 fibroblasts. Here, we further examined the possible mechanism of this ethanol action. Ethanol (10-300 mM) and the protein kinase C (PKC) activator 12-O-tetradecanoyl-phorbol 13-acetate (TPA) had synergistic stimulatory effects on the degradation of preformed [14C]PtdEtn when added in combination to [14C]ethanolamine-labelled suspended NIH 3T3 cells 30 min after collection of cells by scraping. Scraping caused a transient increase, lasting for less than 30 min, in the cellular content of 1,2-diacylglycerol, another PKC activator. Initially (0-50 min incubation), the main water-soluble product of [14C]PtdEtn degradation in ethanol plus TPA-treated cells was [14C]ethanolamine, while later (90 min) the main product of [14C]PtdEtn hydrolysis was [14C]ethanolamine phosphate in the presence of these agents. Ethanol also potentiated the specific stimulatory effects of sphingosine (through phospholipase D) and 4-hydroxynonenal (not involving phospholipase D) on PtdEtn hydrolysis. The effects of these latter agents were unrelated to PKC activation. These data indicate that the observed potentiating effects of ethanol on PtdEtn hydrolysis do not involve direct regulation of PKC or phospholipase D activities.     

Protein kinase C-stimulated formation of ethanolamine from phosphatidylethanolamine involves a protein phosphorylation mechanism: negative regulation by p21 Ras protein

Mammalian cells express a phospholipase D (PLD)-like enzyme which forms ethanolamine from phosphatidylethanolamine (PtdEtn) by a protein kinase C-alpha (PKC-alpha)-activated, presently unknown, mechanism. Now we report that addition of a PKC-alpha-enriched purified PKC preparation or recombinant PKC-alpha to a plasma membrane-enriched membrane fraction, isolated from leukemic HL60 cells, greatly ( approximately 6.5-fold stimulation) enhanced PtdEtn hydrolysis if the PKC activator phorbol 12-myristate 13-acetate (PMA) and ATP were both present; this was accompanied by PKC-mediated phosphorylation of several membrane proteins. The combined effects of PKC-alpha, ATP, and PMA on [(14)C]PtdEtn hydrolysis were inhibited by GF 109203X (10 microM), an inhibitor of catalytic activity of PKC. In this membrane fraction, PMA alone also had a smaller ( approximately 3.5-fold) stimulatory effect on PtdEtn hydrolysis which was not affected by adding ATP or GF 109203X to the membranes. These results suggest that PMA can stimulate PtdEtn hydrolysis via a PKC-catalyzed phosphorylation mechanism as well as by a phosphorylation-independent process. Transformation of NIH 3T3 fibroblasts by H-ras reduced the effect of PMA on PtdEtn hydrolysis. Furthermore, in NIH 3T3 fibroblasts, scrape-loaded Y13-259 anti Ras antibody enhanced PMA-stimulated hydrolysis of PtdEtn. These results suggest that activation of the PtdEtn-hydrolyzing PLD enzyme by PKC-alpha is inhibited by p21 Ras.     

Sphingosine can pre- and post-condition heart and utilizes a different mechanism from sphingosine 1-phosphate

Consistent with previous reports, sphingosine at a high concentration (5 microM) was cardiotoxic as evidenced by increased infarct size in response to ischemia/reperfusion in an ex vivo rat heart. Sphingosine 1-phosphate (S1P) at 5 microM was cardioprotective. However, at a physiologic concentration (0.4 microM) sphingosine as well as S1P was effective in protecting the heart from ischemia/reperfusion injury both when perfused prior to 40 min of ischemia (preconditioning) or when added to reperfusion media following ischemia (postconditioning). Protection by sphingosine and S1P was evidenced with both pre- and post-conditioning by a >75% recovery of left ventricular developed pressure during reperfusion and a decrease in infarct size from 45% of the risk area to less than 8%. When VPC23019, an S1P(1and3)G-protein coupled receptor antagonist, was added to the preconditioning or postconditioning medium along with S1P, it completely blocked S1P-induced protection. However, VPC 23019 did not affect the ability of 0.4 microM sphingosine to either precondition or postcondition hearts. Studies of preconditioning revealed that inhibition of protein kinase C with GF109203X blocked preconditioning by S1P. However, GF109203X did not affect preconditioning by 0.4 microM sphingosine. Likewise, cotreatment with the PI3 kinase inhibitor wortmanin blocked preconditioning by S1P but not by sphingosine. By contrast, inhibition of protein kinase G with KT5823 had no effect on S1P preconditioning but completely eliminated preconditioning by sphingosine. Also, the protein kinase A inhibitory peptide 14-22 amide blocked preconditioning by sphingosine but not S1P. These data reveal for the first time that sphingosine is not toxic at physiologic concentrations but rather is a potent cardioprotectant that utilizes a completely different mechanism than S1P; one that is independent of G-protein coupled receptors and utilizes cyclic nucleotide-dependent pathways.     

Receptor-mediated activation of phospholipase D by sphingosine 1-phosphate in skeletal muscle C2C12 cells. A role for protein kinase C.

The present study showed that sphingosine 1-phosphate (SPP) induced rapid stimulation of phospholipase D (PLD) in skeletal muscle C2C12 cells. The effect was receptor-mediated since it was fully inhibited by pertussis toxin. All known SPP-specific receptors, Edg-1, Edg-3 and AGR16/H218, resulted to be expressed in C2C12 myoblasts, although at a different extent. SPP-induced PLD activation did not involve membrane translocation of PLD1 or PLD2 and appeared to be fully dependent on protein kinase C (PKC) catalytic activity. SPP increased membrane association of PKCalpha, PKCdelta and PKClambda, however, only PKCalpha and PKCdelta played a role in PLD activation since low concentrations of GF109203X and rottlerin, a selective inhibitor of PKCdelta, prevented PLD stimulation.     

Protein kinase C theta and epsilon promote T-cell survival by a rsk-dependent phosphorylation and inactivation of BAD

Both MAPK and protein kinase C (PKC) signaling pathways promote cell survival and protect against cell death. Here, we show that 12-O-tetradecanoylphorbol-13-acetate (TPA) prevents Fas-induced apoptosis in T lymphocytes. The effect of TPA was specifically abolished by the PKC inhibitor GF109203X and by dominant negative PKCtheta, PKCepsilon, and PKCalpha, suggesting that novel and conventional PKC isoforms mediate phorbol ester action. Moreover, TPA stimulated phosphorylation of BAD at serine 112, an effect abrogated by GF109203X but not by the MEK inhibitor PD98059. Expression of constitutively active PKC increased the phosphorylation of BAD at serine 112 but not at serine 136. Additionally, Fas-mediated cell death was enhanced by overexpression of a catalytically inactive form of p90Rsk (Rsk2-KN). Finally, Rsk2-KN abolished the protective effect of constitutively active PKC and totally blocked phosphorylation of BAD on serine 112. Thus, novel PKCtheta and PKCepsilon rescue T lymphocytes from Fas-mediated apoptosis via a p90Rsk-dependent phosphorylation and inactivation of BAD.     

The P2X(7) receptor-mediated phospholipase D activation is regulated by both PKC-dependent and PKC-independent pathways in a rat brain-derived Type-2 astrocyte cell line, RBA-2.

The aim of this study was to characterize the regulatory mechanisms of the P2X(7) receptor (P2X(7)R)-mediated phospholipase D (PLD) activation in a rat brain-derived Type-2 astrocyte cell line, RBA-2. A time course study revealed that activation of P2X(7)R resulted in a choline and not phosphorylcholine formation, suggesting that activation of P2X(7)R is associated with the phosphatidylcholine-PLD (PC-PLD) in these cells. GF 109203X, a selective protein kinase C (PKC) inhibitor, partially inhibited the P2X(7)R-mediated PLD activation, while blocking the phorbol 12-myristate 13-acetate (PMA)-stimulated PLD activity. In addition, PMA synergistically activated the P2X(7)R-mediated PLD activity. Furthermore, genistein, a tyrosine kinase inhibitor, blocked the P2X(7)R-activated PLD, while KN62, a Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor, was less effective, whereas the mitogen-activated protein kinase (MAPK) inhibitor PD98059 was ineffective. No additive inhibitory effects were found by simultaneous treatment of GF 109203X and KN62 on P2X(7)R-activated PLD. Taken together, these results demonstrate that both PKC-dependent and PKC-independent signaling pathways are involved in the regulation of P2X(7)R-mediated PLD activation. Additionally, CaMKII may participate in the PKC-dependent pathway, and tyrosine kinase may play a pivotal role on both PKC-dependent and PKC-independent pathways in the P2X(7)R-mediated PLD activation in RBA-2 cells.     

Role of protein kinase C delta in reactivation of Kaposi's sarcoma-associated herpesvirus

TPA (12-O-tetradecanoylphorbol-13-acetate), a well-known activator of protein kinase C (PKC), can experimentally induce reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) in certain latently infected cells. We selectively blocked the activity of PKC isoforms by using GF 109203X or rottlerin and demonstrated that this inhibition largely decreased lytic KSHV reactivation by TPA. Translocation of the PKCdelta isoform was evident shortly after TPA stimulation. Overexpression of the dominant-negative PKCdelta mutant supported an essential role for the PKCdelta isoform in virus reactivation, yet overexpression of PKCdelta alone was not sufficient to induce lytic reactivation of KSHV, suggesting that additional signaling molecules participate in this pathway.     

Involvement of different protein kinases and phospholipases A2 in phorbol ester (TPA)-induced arachidonic acid liberation in bovine platelets

The effect of various phospholipase A2 and protein kinase inhibitors on the arachidonic acid liberation in bovine platelets induced by the protein kinase activator 12-O-tetradecanoylphorbol-13-acetate (TPA) was studied. TPA stimulates arachidonic acid release mainly by activating group IV cytosolic PLA2 (cPLA2), since inhibitors of this enzyme markedly inhibited arachidonic acid formation. However, group VI Ca2+-independent PLA2 (iPLA2) seems to contribute to the arachidonic acid liberation too, since the relatively specific iPLA2 inhibitor bromoenol lactone (BEL) decreased arachidonic acid generation in part. The pronounced inhibition of the TPA-induced arachidonic acid release by the protein kinase C (PKC) inhibitors GF 109203X and Ro 31-82220, respectively, and by the p38 MAP kinase inhibitor SB 202190 suggests that the activation of the PLA2s by TPA is mediated via PKC and p38 MAP kinase.     

Phorbol ester stimulates the hydrolysis of phosphatidylethanolamine in leukemic HL-60, NIH 3T3, and baby hamster kidney cells

Treatment of leukemic HL-60, NIH 3T3, and baby hamster kidney (BHK-21) cells, prelabeled with [2-14C]ethanolamine, with 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent activator of protein kinase C, resulted in increased degradation of both 14C-labeled phosphatidylethanolamine and its alkenyl (plasmalogen) derivate. A half-maximal and a maximal (approximately 3.4-fold) stimulation of ethanolamine phospholipid degradation required 3 and 10-20 nM TPA, respectively. TPA had a similar concentration-dependent stimulatory effect on the hydrolysis of phosphatidylcholine in cells previously prelabeled with [methyl-14C]choline. Increased phospholipid degradation was not accompanied by the formation of lysophosphatidylethanolamine, indicating that a phospholipase A-type enzyme was not involved. About 80% of total water-soluble degradation products was ethanolamine, suggesting that phospholipid hydrolysis was catalyzed by a phospholipase D-type enzyme. Increased formation of ethanolamine with exposure of cells to TPA was observed only after a 10-min lag period. Mezerein, bryostatin, sn-1-oleoyl-2-acetylglycerol, and polymyxin B, all of which mimic the action of TPA on protein phosphorylation in vivo, also stimulated the hydrolysis of ethanolamine phospholipids in HL-60 cells, suggesting that the TPA effect was mediated by protein kinase C.     

Regulation of ERK1/2 by the C. elegans muscarinic acetylcholine receptor GAR-3 in Chinese hamster ovary cells

Three G-protein-linked acetylcholine receptors (GARs) exist in the nematode C. elegans. GAR-3 is pharmacologically most similar to mammalian muscarinic acetylcholine receptors (mAChRs). We observed that carbachol stimulated ERK1/2 activation in Chinese hamster ovary (CHO) cells stably expressing GAR-3b, the predominant alternatively spliced isoform of GAR-3. This effect was substantially reduced by the phospholipase C (PLC) inhibitor U73122 and the protein kinase C (PKC) inhibitor GF109203X, implying that PLC and PKC are involved in this process. On the other hand, GAR-3b-mediated ERK1/2 activation was inhibited by treatment with forskolin, an adenylate cyclase (AC) activator. This inhibitory effect was blocked by H89, an inhibitor of cAMP-dependent protein kinase A (PKA). These results suggest that GAR-3b-mediated ERK1/2 activation is negatively regulated by cAMP through PKA. Together our data show that GAR-3b mediates ERK1/2 activation in CHO cells and that GAR-3b can couple to both stimulatory and inhibitory pathways to modulate ERK1/2.     

TPA induces the expression of EC-SOD in human monocytic THP-1 cells: involvement of PKC, MEK/ERK and NOX-derived ROS

Extracellular-superoxide dismutase (EC-SOD) is a major SOD isozyme mainly present in the vascular wall. EC-SOD is also observed in monocytes/macrophages, and its high expression contributes to the suppression of atherosclerosis by scavenging superoxide. The molecular mechanisms governing cell-specific expression of EC-SOD are mostly unknown, while the anti-oxidative effect of EC-SOD is well recognized. In this study, we investigated the expression of EC-SOD during 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced monocytic differentiation of THP-1 cells, which is not expressing its gene in the basal phase. We confirmed the significant induction of EC-SOD in a TPA time-dependent manner, and that induction was completely blocked by pre-treatment with GF109203X, an inhibitor of protein kinase C, U0126 and PD98059, inhibitors of mitogen-activated protein kinase kinase/extracellular-signal regulated kinase. Moreover, we determined the involvement of NADPH oxidase-derived reactive oxygen species in that induction. Overall, we considered that these results may contribute to clarify the cell-specific expression of EC-SOD.     

Gonadotropin-releasing hormone activates phospholipase D in ovarian granulosa cells. Possible role in signal transduction

We have investigated the stimulation of phospholipase D activity by the gonadotropin-releasing hormone receptor agonist [D-Ala6, des-Gly10]GnRH N-ethylamide (GnRH-A) in preovulatory, cultured granulosa cells. GnRH-A stimulated up to 10-fold accumulation of phosphatidylethanol, produced by phospholipase D phosphatidyl transferase activity when ethanol acts as the phosphatidyl group acceptor. The effect of GnRH-A was concentration dependent (EC50 = 1 nM) and was inhibited by a specific GnRH receptor antagonist. Low GnRH-A concentrations (less than 10 nM) stimulated also accumulation of phosphatidic acid, but at higher concentrations this response was attenuated. Propranolol, which inhibits phosphatidic acid phosphohydrolase, increased both basal and GnRH-A-stimulated production of phosphatidic acid. A protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA, 100 nM), increased up to 30-fold phosphatidylethanol levels. The effects of supramaximal concentrations of GnRH-A (50 nM) and TPA (1 microM) on the accumulation of phosphatidylethanol were additive, suggesting that the two agents may not act via the same mechanism. This is supported by the fact that 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, a protein kinase C inhibitor, inhibited the effect of TPA 50%, but not that of GnRH-A. However, 24 h pretreatment with TPA abolished cellular response to subsequent treatment with either TPA or GnRH-A. The stimulatory action of GnRH on steroidogenesis could be mimicked by elevating endogenous phosphatidic acid levels in granulosa cells. Exogenous phospholipase D (from Streptomyces chromofuscus, 10 IU/ml) significantly increased (2.7-fold) progesterone production by the cells; under the same conditions, GnRH-A and FSH stimulated progesterone production 3- and 2.6-fold, respectively. Similarly, propranolol stimulated progesterone production 2.2-fold. These results suggest that, in granulosa cells, GnRH receptors are coupled to a phospholipase D whose activation may participate in transducing the GnRH signal for accelerated steroidogenesis. Phospholipase D activity can be independently regulated also by protein kinase C. The possible interrelationships between phospholipase D and other phospholipases which may be activated by GnRH in these ovarian cells are discussed.     

TPA induces the expression of EC-SOD in human monocytic THP-1 cells: Involvement of PKC,MEK/ERK and NOX-derived ROS

Abstract

Extracellular-superoxide dismutase (EC-SOD) is a major SOD isozyme mainly present in the vascular wall. EC-SOD is also observed in monocytes/macrophages, and its high expression contributes to the suppression of atherosclerosis by scavenging superoxide. The molecular mechanisms governing cell-specific expression of EC-SOD are mostly unknown, while the anti-oxidative effect of EC-SOD is well recognized. In this study, we investigated the expression of EC-SOD during 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced monocytic differentiation of THP-1 cells, which is not expressing its gene in the basal phase. We confirmed the significant induction of EC-SOD in a TPA time-dependent manner, and that induction was completely blocked by pre-treatment with GF109203X, an inhibitor of protein kinase C, U0126 and PD98059, inhibitors of mitogen-activated protein kinase kinase/extracellular-signal regulated kinase. Moreover, we determined the involvement of NADPH oxidase-derived reactive oxygen species in that induction. Overall, we considered that these results may contribute to clarify the cell-specific expression of EC-SOD.     

ATP stimulates glucose transport through activation of P2 purinergic receptors in C(2)C(12) skeletal muscle cells

Extracellular ATP acts as a signal that regulates a variety of cellular processes via binding to P2 purinergic receptors (P2 receptors). We herein investigated the effects and signaling pathways of ATP on glucose uptake in C(2)C(12) skeletal muscle cells. ATP as well as P2 receptor agonists (ATP-gamma S) stimulated the rate of glucose uptake, while P2 receptor antagonists (suramin) inhibited the stimulatory effect of ATP, indicating that P2 receptors are involved. This ATP-stimulated glucose transport was blocked by specific inhibitors of Gi protein (pertusiss toxin), phospholipase C (U73122), protein kinase C (GF109203X), and phosphatidylinositol (PI) 3-kinase (LY294002). ATP stimulated PI 3-kinase activity and P2 receptor antagonists blocked this activation. In C(2)C(12) myotubes expressing glucose transporter GLUT4, ATP increased basal and insulin-stimulated glucose transport. Finally, ATP facilitated translocation of GLUT1 and GLUT4 into plasma membrane. These results together suggest that cells respond to extracellular ATP to increase glucose transport through P2 receptors.     

Sphingosine 1-phosphate activates Erk-1/-2 by transactivating epidermal growth factor receptor in rat-2 cells

We investigated a signaling pathway leading to activation of extracellular signal-regulated protein kinase (Erk) 1 and 2 in Rat-2 cells stimulated with sphingosine 1-phosphate (S1P). S1P treatment transiently activated Erk-1/-2 in a dose-dependent manner, and its activation was blocked by pertussis toxin, expression of RasN17, or inhibition of Raf or MEK-1/-2. S1P-induced activation of Erk-1/-2 was also suppressed by the inhibition of epidermal growth factor (EGF) receptor tyrosine kinase with the specific inhibitor AG1478, suggesting that activation of EGF receptor tyrosine kinase was involved in the signaling pathway. S1P-induced Erk-1/-2 activation was enhanced up to 2-fold by inhibiting protein kinase C (PKC) with GF109203X, and PKC inhibition in the absence of S1P treatment also activated Erk-1/-2. The stimulatory effects of Erk-1/-2 activation by PKC inhibition was blocked by treating cells with AG1478, suggesting the involvement of PKC in the regulation of EGF receptor tyrosine kinase activation that leads to Erk-1/-2 activation. Together, these results suggest that S1P activates the EGF receptor through a PKC-dependent pathway that links Ras signaling to the activation of Erk-1/-2 in Rat-2 cells.     

Dual regulation of phospholipase D1 by protein kinase C alpha in vivo

The regulation of phospholipase D1 (PLD1), which has been shown to be activated by protein kinase C (PKC) alpha, was investigated in the human melanoma cell lines. In G361 cell line, which lacks PKCalpha, 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced PLD activation was potentiated by introducing PKCalpha by the adenovirus vector. The kinase-negative PKCalpha elevated TPA-induced PLD activity less significantly than the wild type. A PKC specific inhibitor GF109203X lowered PLD activation in the cells expressing PKCalpha, but did not prevent PLD potentiation induced by the kinase-negative PKCalpha. Expression of PKCbetaII and the kinase-negative PKCbetaII enhanced TPA-stimulated PLD activity moderately in MeWo cell line, in which PKCbetaII is absent. Furthermore, the TPA treatment increased the association of PKCalpha, PKCbetaII, and their kinase-negative mutants with PLD1 in melanoma cells. These results indicate that PLD1 is dually regulated through phosphorylation as well as through the protein-protein interaction by PKCalpha, and probably by PKCbetaII, in vivo.     

ATP stimulates the hydrolysis of phosphatidylethanolamine in NIH 3T3 cells. Potentiating effects of guanosine triphosphates and sphingosine

Recently, phospholipase D-mediated hydrolysis of phosphatidylethanolamine (PtdEtn) was shown to be stimulated by activators of protein kinase C (Kiss, Z., and Anderson, W. B. (1989) J. Biol. Chem. 264, 1483-1487), suggesting that PtdEtn metabolism may play a role in signal transduction. Here we have studied the possible regulation of PtdEtn hydrolysis by adenine and guanine nucleotides, as well as by sphingosine, both in membranes isolated from [14C]ethanolamine- or [32P]PtdEtn-prelabeled NIH 3T3 cells and in intact cells. In isolated membranes both ATP and ADP stimulated the hydrolysis of PtdEtn. Both nucleotides had maximal (approximately 2-fold) effects at about 0.5 mM concentration. The main water-soluble product of [14C]PtdEtn hydrolysis was [14C]ethanolamine, while in [32P] PtdEtn-prelabeled membranes the nucleotides stimulated the formation of [32P]phosphatidic acid, suggesting the involvement of a phospholipase D-type enzyme. The hydrolysis-resistant analogs of GTP, such as guanosine 5'-3-O-(thio)triphosphate and guanyl-5'-yl imidodiphosphate, greatly potentiated the stimulatory effects of ATP and ADP on PtdEtn hydrolysis. On the other hand, the nonphosphorylating analogs of ATP, adenyl-5'-yl beta,gamma-imidodiphosphate and beta,gamma-methyl-eneadenosine 5'-triphosphate, failed to stimulate PtdEtn hydrolysis both in the absence and presence of guanosine triphosphates. Sphingosine, while exhibiting no effect alone, had a relatively modest (1.2-1.3-fold) potentiating effect on ATP-stimulated PtdEtn hydrolysis in isolated membranes. The effect of sphingosine was mimicked by threo- and erythrosphinganines, while N-acetylsphingosine was without effect. In studies with [14C]ethanolamine-prelabeled intact NIH 3T3 cells, externally added ATP did not stimulate PtdEtn hydrolysis. In contrast, sphingosine and sphinganines had much greater stimulatory effects on PtdEtn hydrolysis in intact cells than with isolated membranes. These data indicate that PtdEtn hydrolysis may be regulated by adenine and guanine nucleotides in addition to, or in cooperation with, the activators of protein kinase C, and that sphingosine may be an additional regulator of PtdEtn hydrolysis.