Phorbol 12-myristrate 13-acetate Research Articles

Cellular response of THP-1 macrophages to polystyrene microplastics exposure

Microplastic in the environment have the capability to reach the human immune system via the ingestion, inhalation and direct contact. Polystyrene (PS) is one of the most widely used plastics, which is made up by polymerization of styrene monomers. Mounting evidences on the presence of microplastics in blood clearly indicate their access to macrophages that are major component of the immune system. However, data on the response of macrophages to microplastics exposure are limited. Our study reports the response of human macrophages transformed by PMA (phorbol 12-myristrate 13-acetate) to exposure to PSNPs of size range (≤ 450 nm). The polystyrene particles utilized in this study, were formulated from beads to powder by grinding and filtering the particles to acquire a range of size ≤ 450 nm particles with deionized water. This size variation used in this experiment imitates the size of plastic that humans can ingest plastic particles through food that gets fragmented from plastic cups and plates. Here we report that exposure to PSNPs (50–500 µg/mL) significantly decreased the viability of human macrophages. In addition, PSNPs (500 µg/mL) induced oxidative stress and decrease cell proliferation. Exposure to PSNPs decrease the membrane potential of mitochondria and caused damage to the DNA of macrophages. Overall, our study reports the differential toxic effects of PSNPs on human macrophages, delineating the potential risks of PSNPs exposure to human health.

Lipoteichoic acid of Staphylococcus aureus enhances IL-6 expression in activated human basophils

At allergic inflammation, cross-linking of FcɛRI with multivalent antigen-bound IgE triggers the signaling pathways via activation of protein kinase C (PKC) and mobilization of intracellular Ca(2+) leading to the production of various mediators such as interleukin-6 (IL-6). Accumulating reports demonstrated that interaction of Toll-like receptor 2 (TLR2) expressed on basophils with a TLR2 ligand, lipoteichoic acid (LTA) of Staphylococcus aureus, exacerbated allergic inflammation. Here, we showed that staphylococcal LTA (Sa.LTA) substantially enhanced IL-6 expression at both the protein and the mRNA levels in the human basophil line, KU812, in the presence of a PKC activator (phorbol 12-myristrate 13-acetate; PMA), and a calcium ionophore (A23187), whereas Sa.LTA alone could not induce IL-6 expression. PMA/A23187 augmented the expression of CD14 and TLR2 on the surface of KU812 cells and concomitantly increased the binding of fluorochrome-labeled Sa.LTA to the cells. Sa.LTA enhanced the phosphorylation of mitogen-activated protein (MAP) kinases in PMA/A23187-stimulated KU812 cells. Notably, Sa.LTA could not enhance PMA/A23187-induced IL-6 expression in the presence of inhibitors of MAP kinases, reactive oxygen species, or protein kinase C. Furthermore, Sa.LTA enhanced the PMA/A23187-increased DNA-binding activities of the transcription factors NF-κB and AP-1. Experiments using human peripheral blood mononuclear cells demonstrated that not only PMA/A23187 but also Sa.LTA increased the intracellular IL-6 expression in the basophil population and Sa.LTA plus PMA/A23187 further enhanced the IL-6 expression. Collectively, these results suggest that Sa.LTA exacerbates allergic inflammation by potentiating IL-6 production from activated basophils.

Phosphorylation of TRPC6 Channels at Thr69 Is Required for Anti-hypertrophic Effects of Phosphodiesterase 5 Inhibition

Activation of Ca(2+) signaling induced by receptor stimulation and mechanical stress plays a critical role in the development of cardiac hypertrophy. A canonical transient receptor potential protein subfamily member, TRPC6, which is activated by diacylglycerol and mechanical stretch, works as an upstream regulator of the Ca(2+) signaling pathway. Although activation of protein kinase G (PKG) inhibits TRPC6 channel activity and cardiac hypertrophy, respectively, it is unclear whether PKG suppresses cardiac hypertrophy through inhibition of TRPC6. Here, we show that inhibition of cGMP-selective PDE5 (phosphodiesterase 5) suppresses endothelin-1-, diacylglycerol analog-, and mechanical stretch-induced hypertrophy through inhibition of Ca(2+) influx in rat neonatal cardiomyocytes. Inhibition of PDE5 suppressed the increase in frequency of Ca(2+) spikes induced by agonists or mechanical stretch. However, PDE5 inhibition did not suppress the hypertrophic responses induced by high KCl or the activation of protein kinase C, suggesting that PDE5 inhibition suppresses Ca(2+) influx itself or molecule(s) upstream of Ca(2+) influx. PKG activated by PDE5 inhibition phosphorylated TRPC6 proteins at Thr(69) and prevented TRPC6-mediated Ca(2+) influx. Substitution of Ala for Thr(69) in TRPC6 abolished the anti-hypertrophic effects of PDE5 inhibition. In addition, chronic PDE5 inhibition by oral sildenafil treatment actually induced TRPC6 phosphorylation in mouse hearts. Knockdown of RGS2 (regulator of G protein signaling 2) and RGS4, both of which are activated by PKG to reduce G alpha(q)-mediated signaling, did not affect the suppression of receptor-activated Ca(2+) influx by PDE5 inhibition. These results suggest that phosphorylation and functional suppression of TRPC6 underlie prevention of pathological hypertrophy by PDE5 inhibition.

Anti-inflammatory effect of Equisetum hyemale via suppression of nuclear factor-κB activation in human mast cells

Equisetum hyemale Linne. (EH) (Equisetaceae) has been used for the treatment of eye and skin disease, chronic eczema, pneumoconiosis and asthma in Korea and China. Human leukemic mast cells are widely distributed in the connective tissues of mammals and other vertebrates. Phorbol 12-myristrate 13-acetate (PMA) and calcium ionophore A23187 stimulated Human leukaemic mast cell line-1 (HMC-1) can produce a variety of inflammatory mediators and several pro-inflammatory and chemotactic cytokines such as TNF-<TEX>$\alpha$</TEX>, IL-6 and IL-8. Since TNF-<TEX>$\alpha$</TEX>, IL-6 and IL-8 are major factors during the inflammatory process, we studied the effects of EH on TNF-<TEX>$\alpha$</TEX>, IL-6 and IL-8 release in HMC-1 stimulated with PMA and A23187. The result of this study indicate that EH inhibits TNF-<TEX>$\alpha$</TEX>, IL-6 and IL-8 in activated HMC-1 cells via <TEX>$I{\kappa}B$</TEX>/Nuclear factor-<TEX>${\kappa}B$</TEX> pathway. Therefore, EH might contribute significantly to the prevention or treatment of mast-cell mediated inflammatory diseases and EH has potential use in the therapy of chronic allergic inflammation.

PKC-dependent phosphorylations modify the action of deltamethrin on rat brain N-type (Ca V2.2) voltage-sensitive calcium channel

Voltage clamp electrophysiological studies using wild type Ca V2.2 and its β 3 subunit coexpressed in Xenopus oocytes revealed that deltamethrin increased the rate of activation, prolonged inactivation and reduced peak current. Site-directed mutagenesis of threonine 422 to glutamic acid (T422E, one of five protein kinase C (PKC)-dependent phosphorylation sites) resulted in a channel that acted as if it were permanently phosphorylated. This resulted in an increased probability of opening during depolarization and a reduced inhibition by the βγ subunit of heterotrimeric G-protein. Deltamethrin treatment of T422E Ca V2.2 enhanced peak current ∼50% over ethanol-treated controls with an EC 50 of 9.8 × 10 −11 M. Phosphorylation of wild type Ca V2.2 is evoked by the phorbol ester, phorbol 12-myristrate, 13 acetate (PMA), by activating endogenous protein kinase C (PKC) in oocytes. PKC-dependent phosphorylation activated by PMA of wild type Ca V2.2 has been previously shown to slow channel deactivation and increased Ca 2+ influx and subsequent neurotransmitter release. Following PMA-activated phosphorylation, deltamethrin significantly increased peak current and slowed deactivation of the phosphorylated channel, which would be consistent with slower channel closure, greater Ca 2+ influx and enhanced neurotransmitter release seen in vivo. Deltamethrin treatment in the absence of PMA-activated phosphorylation resulted in no effect on the deactivation kinetics of unphosphorylated Ca V2.2 or the T422E mutant. Thus, Ca V2.2 is modified by deltamethrin but the resulting perturbations are dependent upon its PKC-dependent phosphorylation state.

Efflux of sphingomyelin, cholesterol, and phosphatidylcholine by ABCG1

Cholesterol and phospholipids are essential to the body, but an excess of cholesterol or lipids is toxic and a risk factor for arteriosclerosis. ABCG1, one of the half-type ABC proteins, is thought to be involved in cholesterol homeostasis. To explore the role of ABCG1 in cholesterol homeostasis, we examined its subcellular localization and function. ABCG1 and ABCG1-K120M, a WalkerA lysine mutant, were localized to the plasma membrane in HEK293 cells stably expressing ABCG1 and formed a homodimer. A stable transformant expressing ABCG1 exhibited efflux of cholesterol and choline phospholipids in the presence of BSA, and the cholesterol efflux was enhanced by the presence of HDL, whereas cells expressing ABCG1-K120M did not, suggesting that ATP binding and/or hydrolysis is required for the efflux. Mass and TLC analyses revealed that ABCG1 and ABCA1 secrete several species of sphingomyelin (SM) and phosphatidylcholine (PC), and SMs were preferentially secreted by ABCG1, whereas PCs were preferentially secreted by ABCA1. These results suggest that ABCA1 and ABCG1 mediate the lipid efflux in different mechanisms, in which different species of phospholipids are secreted, and function coordinately in the removal of cholesterol and phospholipids from peripheral cells.

Comparison study of the expressions of myristoylated alanine-rich C kinase substrate in hepatocellular carcinoma, liver cirrhosis, chronic hepatitis, and normal liver

The myristoylated alanine-rich C kinase substrate (MARCKS) is a prominent substrate for protein kinase C (PKC) in a variety of cells. The aim of this study was not only to evaluate the expression and localization of MARCKS in various pathological liver tissues, including HCC, but also to analyze the difference in MARCKS expression between hepatitis virus-induced HCC and cirrhosis. The level of MARCKS and its phosphorylated proteins, as well as its localization, were determined using Western blot and/or immunohistochemistry in HCC and other pathological liver tissues. We also analyzed the change of MARCKS localization on the influence of MARCKS phosphorylation in the HLF cancer cell line by phosphorylation study. In addition, the relationship between MARCKS expression and proliferative activity was studied in HCC. In the immunohistochemical study, a very small amount of MARCKS protein was found along the contour of the hepatocellular membrane in normal liver and in cases of chronic hepatitis. MARCKS was up-regulated in liver cirrhosis tissue and was localized in the cytoplasm of hepatocytes. The expression of MARCKS was down-regulated in HCC tissues, as compared with non-tumorous liver cirrhosis tissues from the same patients. Furthermore, MARCKS was serine-phosphorylated in liver cirrhosis and HCC, and phosphorylated MARCKS was detected in a cytosolic fraction of these tissues. In a phosphorylation study using the HLF HCC cell line, MARCKS was displaced from the plasma membrane to the cytosol following the activation of protein kinase C (PKC) by phorbol 12-myristrate 13-acetate (PMA). Furthermore, the activity of cyclin D1 and cyclin E kinases was found to be higher in HCCs with low MARCKS expression than in HCCs with high MARCKS expression. These results suggest that up-regulation of MARCKS might be essential in the generation of cirrhotic nodules through chronic hepatitis from normal liver, and that the phosphorylation and/or down-regulation of MARCKS might play an important role in the development and progression of HCC from liver cirrhosis.

Involvement of high-density lipoprotein in stimulatory effect of hormones supporting function of the bovine corpus luteum.

The hypothesis that epinephrine (noradrenaline, NA) enhances utilisation of high-density lipoproteins (HDL) by bovine luteal cells and that this process involves phospholipase (PL) C and protein kinase (PK) C intracellular pathway was tested. Luteal cells from days 2-4, 5-10 or 11-17 of the oestrous cycle were preincubated for 20 h. Subsequently DMEM/Ham's F-12 medium was replaced by fresh medium and the cells were treated for 6 h as follows: In Experiment I with HDL (5-75 micrograms cholesterol per ml), NA, isoprenaline (ISO) or luteinising hormone (LH). In Experiment II cells were incubated for further 24 h in deficient medium (without FCS) and next treated as in Experiment I. In Experiment III cells were stimulated with NA, ISO or LH alone and together with HDL. In Experiment IV cells were treated with PLC inhibitor (U-73122) or with PKC inhibitor (staurosporine) or stimulator (phorbol 12-myristrate 13-acetate) and with either NA, insulin or LH. Only luteal cells from days 5-10 of the cycle responded on HDL and beta-mimetics (P < 0.05). LH stimulated progesterone secretion from the luteal cells during all stages of the cycle (P < 0.001). Cells incubated in deficient medium and supplemented with HDL secreted as much progesterone as those stimulated by LH in all stages of the cycle. Beta-mimetics were unable to enhance the stimulatory effect of HDL. Blockade of PLC had no influence on progesterone secretion from cells treated with either NA or LH, but this did impair the stimulatory effect of insulin (P < 0.05). Similarly, blockade of PKC by staurosporine impaired (P < 0.05) the effect of insulin only but not that observed after LH or NA treatment. We suggest that: (a) noradrenergic stimulation does not enhance utilisation of cholesterol from HDL for progesterone secretion; (b) the fasting of luteal cells seems to activate enzymes responsible for the progesterone synthesis; (c) effect of NA on progesterone secretion from luteal cells does not involve the PLC-PKC pathway.

Role of PKC isoforms in glucose transport in 3T3-L1 adipocytes: insignificance of atypical PKC.

To elucidate the involvement of protein kinase C (PKC) isoforms in insulin-induced and phorbol ester-induced glucose transport, we expressed several PKC isoforms, conventional PKC-alpha, novel PKC-delta, and atypical PKC isoforms of PKC-lambda and PKC-zeta, and their mutants in 3T3-L1 adipocytes using an adenovirus-mediated gene transduction system. Endogenous expression and the activities of PKC-alpha and PKC-lambda/zeta, but not of PKC-delta, were detected in 3T3-L1 adipocytes. Overexpression of each wild-type PKC isoform induced a large amount of PKC activity in 3T3-L1 adipocytes. Phorbol 12-myristrate 13-acetate (PMA) activated PKC-alpha and exogenous PKC-delta but not atypical PKC-lambda/zeta. Insulin also activated the overexpressed PKC-delta but not PKC-alpha. Expression of the wild-type PKC-alpha or PKC-delta resulted in significant increases in glucose transport activity in the basal and PMA-stimulated states. Dominant-negative PKC-alpha expression, which inhibited the PMA activation of PKC-alpha, decreased in PMA-stimulated glucose transport. Glucose transport activity in the insulin-stimulated state was increased by the expression of PKC-delta but not of PKC-alpha. These findings demonstrate that both conventional and novel PKC isoforms are involved in PMA-stimulated glucose transport and that other novel PKC isoforms could participate in PMA-stimulated and insulin-stimulated glucose transport. Atypical PKC-lambda/zeta was not significantly activated by insulin, and expression of the wild-type, constitutively active, and dominant-negative mutants of atypical PKC did not affect either basal or insulin-stimulated glucose transport. Thus atypical PKC enzymes do not play a major role in insulin-stimulated glucose transport in 3T3-L1 adipocytes.

Acute inhibition of brain-specific Na(+)/H(+) exchanger isoform 5 by protein kinases A and C and cell shrinkage.

Little is known of the functional properties of the mammalian, brain-specific Na(+)/H(+) exchanger isoform 5 (NHE5). Rat NHE5 was stably expressed in NHE-deficient PS120 cells, and its activity was characterized using the fluorescent pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. NHE5 was insensitive to ethylisopropyl amiloride. The transport kinetics displayed a simple Michaelis-Menten relationship for extracellular Na(+) (apparent K(Na) = 27 +/- 5 mM) and a Hill coefficient near 3 for the intracellular proton concentration with a half-maximal activity at an intracellular pH of 6.93 +/- 0.03. NHE5 activity was inhibited by acute exposure to 8-bromo-cAMP or forskolin (which increases intracellular cAMP by activating adenylate cyclase). The kinase inhibitor H-89 reversed this inhibition, suggesting that regulation by cAMP involves a protein kinase A (PKA)-dependent process. In contrast, 8-bromo-cGMP did not have a significant effect on activity. The protein kinase C (PKC) activator phorbol 12-myristrate 13-acetate inhibited NHE5, and the PKC antagonist chelerythrine chloride blunted this effect. Activity was also inhibited by hyperosmotic-induced cell shrinkage but was unaffected by a hyposmotic challenge. These results demonstrate that rat brain NHE5 is downregulated by activation of PKA and PKC and by cell shrinkage, important regulators of neuronal cell function.

PPARγ Ligands Inhibit TNF-α-Induced LOX-1 Expression in Cultured Endothelial Cells

Endothelial dysfunction or activation, elicited by oxidized low-density lipoprotein (OxLDL), has been implicated in the initiation and progression of atherosclerosis. We elucidated whether tumor necrosis factor-α (TNF-α)-induced endothelial OxLDL receptor, lectin-like OxLDL receptor-1 (LOX-1), mRNA expression is modified by peroxisome proliferator-activated receptor (PPAR) activators in cultured bovine aortic endothelial cells (BAEC). We confirmed that both PPARα and PPARγ were expressed in BAEC by reverse transcription-polymerase chain reaction analysis. Natural PPARγ ligand 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) and the thiazolidinediones, pioglitazone and troglitazone, decreased TNF-α-induced LOX-1 mRNA expression in BAEC. LOX-1 expression induced by phorbol 12-myristrate 13-acetate was also inhibited by 15d-PGJ2. In contrast, PPARα ligands, Wy14643 and fenofibric acid, did not alter TNF-α-induced LOX-1 expression. TNF-α-induced immunohistochemical staining of LOX-1 was suppressed by 15d-PGJ2 but not Wy14643. Taken together, PPARγ activators inhibit TNF-α-induced LOX-1 expression in cultured BAEC, which may beneficially influence inflammatory responses in atherosclerosis.

Distribution and regulation of proconvertases PC1 and PC2 in human pituitary adenomas.

Pituitary adenomas are members of the family of neuroendocrine cells and tumors which have secretory granules containing chromogranins/secretogranins and other proteins. Pituitary adenomas express the neuroendocrine specific proconvertases PC1 (also known as PC3) and PC2, which are important for the proteolytic processing of chromogranins/secretogranins molecules. We examined the distribution of PC1 and PC2 in primary cultures of 20 pituitary adenomas and analyzed the regulation of the proconvertase mRNAs and proteins by various secretagogues including hypothalamic hormones and phorbol ester to determine the role of PC1 and PC2 in CgA processing in pituitary adenomas. Although PC2 was present in all adenomas, there was a differential distribution of PC1 with PRL adenomas expressing lower levels of PC1 compared to other adenoma types by RT-PCR analysis, in situ hybridization and immunostaining. Treatment of primary cultures of pituitary adenomas with phorbol 12-myristrate 13-acetate (PMA) resulted in an increase in pancreastatin (PST) secretion in most pituitary adenomas and increased PC1 mRNA and protein expression in gonadotroph adenomas, but not in other types of adenomas. Analysis of a human pituitary adenoma cell line, immortalized by recombinant defective adenovirus (HP75), which expressed chromogranin A, FSH, PC1 and PC2 showed that PST was secreted by these immortalized cells. Treatment with TGF beta 1 resulted in an increase in PST secretion and in PC1 mRNA and protein. These results indicate that a) there is a differential distribution of PC1 in human pituitary adenomas with PRL adenomas expressing very little PC1 mRNA and protein and b) that PC1 expression in gonadotropin hormone-producing adenomas is regulated by PMA and TGF beta 1. These findings support the observation that chromogranin A is a substrate for the endoproteinase PC1 in human pituitary adenoma cells.

Modulation of dopamine transporter functions by protein kinase C

The effects of phosphorylation on the functions of the bovine dopamine transporter (DAT) expressed in CV-1 cells were investigated. The cells expressing DAT stably were prepared by transfection of CV-1 cells (monkey kidney cell line) with plasmid pRc/CMV-DAT which is an expression vector pRc/CMV-based plasmid containing bovine DAT cDNA (Usdin et al., 1991). DAT functional assays followed the preincubations of DAT expressing cells with various modulators of protein kinases, such as phorbol 12-myristrate 13-acetate (PMA), staurosphorin, calphostin C, forskolin, and EGTA, on the concentration- or time-dependent manners. Dopamine uptake into DAT expressing cells was severely decreased by PMA pretreatment, and increased by staurosphorin or calphostin C. Similar effects were observed on the uptakes of 1-methyl-4-phenylpyridinium (MPP+) into DATexpressing cells. However, DAT functions were not altered by forskolin or EGTA. These results suggest that the functions of DAT are modulated by protein kinase C (PKC).

Point mutation in the cytoplasmic domain of the neutrophil p22-phox cytochrome b subunit is associated with a nonfunctional NADPH oxidase and chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a congenital disorder in which phagocytes cannot generate superoxide (O2-) and other microbial oxidants due to mutations in any one of four components of the O2(-)-generating complex, NADPH oxidase. We report here a female CGD patient in whom a missense mutation in one of these components, the p22-phox subunit of the neutrophil membrane cytochrome b [where phox indicates phagocyte oxidase (used to designate protein components of the phagocyte NADPH oxidase)] results in a nonfunctional oxidase and failure of neutrophils to produce O2- in response to phorbol 12-myristrate 13-acetate. Cytochrome b in the patient's neutrophils was normal in appearance and abundance as determined by visible spectroscopy and by immunoblots of the gp91 and p22 subunits. However, the neutrophil plasma membranes were devoid of activity in the cell-free oxidase activation system, whereas the cytosol functioned normally. We postulated that the patient was homozygous for a mutation in p22 that results in the synthesis of normal levels of a nonfunctional cytochrome b. A single-base substitution (C----A) was found in the patient's mononuclear cell p22-phox cDNA that predicts a nonconservative Pro----Gln substitution at residue 156. The same mutation was also identified in all clones sequenced from patient genomic DNA, demonstrating homozygosity for the mutant allele. An antipeptide antibody against p22 residues 153-164 was found to bind only to permeabilized neutrophils, indicating that the mutation occurs in a cytoplasmic domain. These studies establish that this domain of p22-phox is cytoplasmic and that mutations in this region can have profound effects on cytochrome b function.

Possible involvement of adenylyl cyclase-cAMP-protein kinase A pathway in somatostatin inhibition of growth hormone release from chicken pituitary cells

Somatostatin (SRIF) reduces growth hormone releasing hormone (GRF)-stimulated growth hormone (GH) release from avian and mammalian adenohypophyseal cells. The present studies examined the intracellular mechanisms mediating SRIF inhibition of GRF-stimulated GH release from chicken pituitary cells. Increases (P less than 0.05) in GH release were observed in the presence of (1) GRF; (2) the adenylyl cyclase stimulator, forskolin; (3) a cAMP analog, 8-bromo-cAMP; (4) the phosphodiesterase inhibitor 3-isobutyl-l-methyl-xanthine (IBMX) combined with GRF; (5) a tumor-promoting phorbol ester and protein kinase C activator, phorbol 12-myristate, 13-acetate (PMA); (6) a diacylglycerol analog, 1,2-dioctanoyl-glycerol (DiC8); and (7) a calcium ionophore, A23187, alone and in combination with PMA. Somatostatin (10 ng/ml) reduced the release of GH stimulated by GRF, forskolin, and 8-bromo cAMP and the GRF-provoked release of GH in the presence of IBMX (P less than 0.05). Somatostatin, however, did not influence GH release in the presence of the protein kinase C activators, PMA or DiC8, or the calcium ionophore A23187. These data suggest that SRIF inhibits GRF-provoked GH release by reducing the ability of the cAMP-protein kinase A but not of the calcium or protein kinase C intracellular message pathways to stimulate GH release.

Phorbol ester-induced activation of protein kinase C leads to increased formation of diacylglycerol in human neutrophils

Human neutrophils stimulated with a phorbol ester (phorbol 12-myristrate 13-acetate or phorbol 12,13-dibutyrate) responded with an increase in diacylglycerol, considered the natural activator of protein kinase C. The amounts of diacylglycerol formed were considerable, reaching 700–900% of basal after 20 min. In contrast, 4-α-phorbol 12-myristate 13-acetate did not induce any detectable formation of diacylglycerol. Simultaneously, phorbol 12-myristate 13-acetate exposure caused increased breakdown of both phosphatidylcholine and phosphatidylinositol 4,5-bisphosphate. These results suggest that once activated, protein kinase C can positively modulate its own activity by inducing additional formation of diacylglycerol from at least two different sources.