A diacylglycerol kinase is involved in the regulation of interleukin-2 synthesis in Jurkat T cells.

Diacylglycerol (DAG) plays a prominent role in several activation processes because of its ability, in the presence of calcium ions and phosphatidylserine, to activate C kinase. In T lymphocytes, however, DAG, produced in response to activating mAb or lectins, is rapidly metabolized into phosphatidic acid (PA) which may participate in further steps of activation. We thus investigated the involvement of a DAG kinase in several events subsequent to the activation of Jurkat T cells by CD3 mAb or phytohemagglutinin (PHA). We showed that three inhibitors of DAG kinase abrogated PA production and impaired calcium release from intracellular compartment, restored phosphatidylserine synthesis, and finally blocked IL-2 production in CD3- and PHA-stimulated cells. Postactivation DAG levels were not modified and DAG kinase inhibitors lowered IL-2 secretion even in the presence of phorbol ester that directly activates the C kinase. These results clearly demonstrate that, beside the effect of DAG on C kinase, DAG kinase dependent production of PA is crucial for further steps of T cell activation.     

Signalling events mediating the activation of protein kinase C by interleukin-2 in cytotoxic T cells.

Interleukin-2 (IL-2) plays a vital role in the generation and regulation of the immune response, including important aspects of T cell survival. IL-2-mediated survival of T cells appears to be dependent on the activation of a pool of membrane-associated protein kinase C (PKC) that occurs in the absence of detectable translocation of the enzyme from the cytosol to membranes. In this report we investigate the mechanism(s) responsible for this PKC activation after IL-2 stimulation in the cytotoxic T cell line, CTLL-2. Tyrosine kinase activity, activated after IL-2 stimulation, was found not to be linked to the activation of PKC by the cytokine. On the other hand, a pertussis toxin (PTX)-sensitive G protein did appear coupled to PKC activation since PTX effectively blocked IL-2 stimulated PKC activity. Diacylglycerols (DAG), but not inositol 1,3,5-triphosphate (IP3) and intracellular Ca2+, increased after IL-2 stimulation suggesting that DAGs were generated via the phosphatidylcholine-phospholipase C (PC-PLC) or phosphatidylcholine-phospholipase D (PC-PLD) pathways. The increase in DAG by IL-2 was probably necessary for activation of membrane-resident PKC since exogenously applied DAG stimulated this PKC pool in both intact cells and in isolated membranes. IL-2 also increased arachidonic acid (AA) production in CTLL-2 cells, probably via phospholipase A2 (PLA2) since the PLA2 inhibitors oleoyloxyethyl phosphocholine and AACOCF3 (AACF) effectively blocked IL-2 stimulated PKC activation. Exogenous AA also increased PKC activity in intact cells and isolated membranes, suggesting that AA produced by IL-2 receptor stimulation was probably linked to PKC activation. These results suggest that the activation of membrane-resident PKC by IL-2 involves multiple second messengers, including G proteins, DAG and AA.     

Signal transduction through the T cell antigen receptor. Activation of phospholipase C through a G protein-independent coupling mechanism.

The T cell Ag (Ti-CD3) receptor complex has been proposed to regulate phosphoinositide-specific phospholipase C (PLC) through a cholera toxin (CTX)-sensitive guanine nucleotide-binding (G) protein. In this study, we have used CTX and staurosporine as pharmacologic probes to further define the linkage between the Ti-CD3 receptor and PLC activity in the human T cell line, Jurkat. CTX pretreatment inhibited Ti-CD3 receptor-dependent phosphoinositide hydrolysis and, concomitantly, protein tyrosine kinase activation in intact cells. Studies with electrically permeabilized Jurkat cells revealed that guanosine 5'-(3-O-thio) triphosphate stimulated an increase in PLC activity, that unlike the response to Ti-CD3 receptor ligation, was not affected by cellular pretreatment with CTX. In contrast, the phosphotyrosine phosphatase inhibitors, orthovanadate and molybdate anions, stimulated phosphoinositide hydrolysis in permeabilized cells through a CTX-sensitive mechanism of PLC activation. Additional studies with a known PTK inhibitor, staurosporine, supported the results obtained with CTX. Staurosporine pretreatment inhibited the phosphoinositide hydrolysis induced by anti-CD3 antibodies or phosphotyrosine phosphatase inhibitors, but failed to alter the G protein-dependent PLC activation response to guanosine 5'-(3-O-thio) triphosphate. The results of this study indicate that PLC activity(s) in Jurkat cells are regulated by both G protein- and PTK-dependent coupling mechanisms. However, the differential inhibitory effects of CTX and staurosporine on these PLC activation pathways strongly suggest that a protein tyrosine kinase activation event, rather than a G protein, mediates the functional linkage between the Ti-CD3 receptor and PLC activity in Jurkat cells.     

Cyclic AMP can partially restore platelet-derived growth factor-stimulated prostaglandin E2 biosynthesis, and calcium mobilization in EJ-ras-transformed NIH-3T3 cells

NIH-3T3 cells transformed by the EJ-ras oncogene display reduced platelet-derived growth factor (PDGF)-stimulated phospholipase C activity as measured by inositol 1,4,5-triphosphate (IP3) synthesis and Ca2+ mobilization. The lack of PDGF-stimulated Ca2+ mobilization in EJ-ras transformed cells is not due to a loss of IP3 sensitivity, because microinjected IP3 elevates intracellular Ca2+. Treatment of EJ-ras transformed cells with cholera toxin or 8-bromo-cyclic AMP, but not pertussis toxin or the beta-subunit of cholera toxin, results in a slight recovery of PDGF-stimulated IP3 synthesis, a marked increase in intracellular Ca2+ mobilization, and an almost complete recovery of prostaglandin E2 biosynthesis. These data suggest that EJ p21-mediated inhibition of PDGF-stimulated intracellular events can be partially and transiently reversed by cyclic AMP.     

Induction of interleukin-2 receptor-alpha gene expression is regulated by post-translational activation of kappa B specific DNA binding proteins

T cell mitogens induce the expression of specific trans-acting DNA binding proteins that in turn regulate the expression of the interleukin-2 receptor-alpha (IL-2R alpha) gene. To investigate whether de novo protein synthesis is required for the activation of these transacting factors and the induced expression of this receptor gene, Jurkat T cells were incubated with various inhibitors of protein synthesis prior to stimulation with phytohemagglutinin and phorbol 12-myristate 13-acetate (PMA). Despite the presence of cycloheximide or anisomycin at concentrations sufficient to block greater than 97% of cellular protein synthesis, phytohemagglutinin and phorbol 12-myristate 13-acetate effectively induced the expression of the IL-2R alpha gene as measured at the mRNA level. Similarly, gel retardation, DNA footprinting, and DNA-protein cross-linking studies revealed that these mitogens induced the activation of two predominant DNA binding proteins (50-55 and 80-90 kDa) in the presence or absence of cycloheximide and anisomycin. Both of these proteins specifically interacted with a kappa B-like binding site present in the IL-2R alpha promoter (-267 to -256) that is requisite for mitogen-induced expression of this receptor gene. These findings support a post-translational mechanism of induction of pre-existing, but inactive, DNA binding proteins which in turn bind to and activate the IL-2R alpha gene.     

Differential regulation of HSC70, HSP70, HSP90 alpha, and HSP90 beta mRNA expression by mitogen activation and heat shock in human lymphocytes

A subset of heat shock proteins, HSP90 alpha, HSP90 beta, and a member of the HSP70 family, HSC70, shows enhanced synthesis following mitogenic activation as well as heat shock in human peripheral blood mononuclear cells. In this study, we have examined expression of mRNA for these proteins, including the major 70-kDa heat shock protein, HSP70, in mononuclear cells following either heat shock or mitogenic activation with phytohemagglutinin (PHA), ionomycin, and the phorbol ester, tetradecanoyl phorbol acetate. The results demonstrate that the kinetics of mRNA expression of these four genes generally parallel the kinetics of enhanced protein synthesis seen following either heat shock or mitogen activation and provide clear evidence that mitogen-induced synthesis of HSC70 and HSP90 is due to increased mRNA levels and not simply to enhanced translation of preexisting mRNA. Although most previous studies have focused on cell cycle regulation of HSP70 mRNA, we found that HSP70 mRNA was only slightly and transiently induced by PHA activation, while HSC70 is the predominant 70-kDa heat shock protein homologue induced by mitogens. Similarly, HSP90 alpha appears more inducible by heat shock than mitogens while the opposite is true for HSP90 beta. These results suggest that, although HSP70 and HSC70 have been shown to contain similar promoter regions, additional regulatory mechanisms which result in differential expression to a given stimulus must exist. They clearly demonstrate that human lymphocytes are an important model system for determining mechanisms for regulation of heat shock protein synthesis in unstressed cells. Finally, based on kinetics of mRNA expression, the results are consistent with the hypothesis that HSC70 and HSP90 gene expression are driven by an IL-2/IL-2 receptor-dependent pathway in human T cells.     

Differential CD3/T cell antigen receptor-mediated IL-2 production in jurkat T cells. Dissociation of IL-2 response from total inositol phosphate and calcium responses

We used three anti-human anti-CD3 mAb each recognizing different surface CD3 epitopes to differentially perturb the CD3/TCR complex on the surface of Jurkat T cells. In the presence of phorbol ester, these anti-CD3 mAb triggered differential IL-2 production in Jurkat T cells, which could not be explained by differences in kinetics of IL-2 production, by differences in IL-2 adsorption caused by differential surface expression of p55 or p75 IL-2R, by effects on IL-2 secretion rather than actual synthesis, or by differential toxicities of the anti-CD3 mAb to Jurkat cells. In addition, this differential anti-CD3-induced IL-2 production could not be explained by differences in mAb isotype or in avidities of the anti-CD3 mAb for the Jurkat cells. Moreover, anti-CD3 mAb covalently immobilized onto beads also differentially induced IL-2 production in Jurkat cells, suggesting that the differential IL-2 response is not based on differential rates of anti-CD3-induced modulation of Jurkat cell surface CD3. Although differences among the anti-CD3 mAb in the initial rates of binding to Jurkat cell were observed, this was also believed unlikely to explain the differential IL-2 response. Regardless of the anti-CD3 mAb used, anti-CD3-induced total inositol phosphate (IP) production did not necessarily correlate with anti-CD3-induced IL-2 production. Nevertheless, despite the differences among the anti-CD3 mAb in inducing IL-2 production, the calcium responses were grossly similar. Taken together, these observations indicate that CD3/TCR-mediated IL-2 production in Jurkat cells can be dissociated from total IP generation, and the basis of differential CD3/TCR-mediated IL-2 production in these cells does not appear to be at the level of the initial activation-induced calcium response. These studies suggest that the nature of the CD3/TCR ligand (its physical form and/or the specific epitope it perturbs) can either directly influence intracellular events distal to the generation of IP and increase in intracellular free calcium leading to differential IL-2 production or can trigger IP-independent pathways that affect IL-2 production.     

Multiple signal transduction pathways lead to extracellular ATP-stimulated mitogenesis in mammalian cells: I. Involvement of protein kinase C-dependent and -independent pathways

We recently reported that extracellular ATP was mitogenic for Swiss 3T3, 3T6, and A431 cells (Huang et al.: Proc. Natl. Acad. Sci. USA, 86:7904-7908, 1989). Here we examined the possible involvement of activation of the protein kinase C (PKC) signal transduction pathway in the mechanism of action of extracellular ATP. A potent synergistic stimulation of DNA synthesis in quiescent cultures of 3T3 and 3T6 cells was observed when ATP was presented in combination with growth factors that activate PKC, such as bombesin, vasopressin, or tumor-promoting phorbol esters. This finding suggests that ATP and these mitogens do not act through a common mechanism. In contrast, ATP was unable to show synergism with phorbol esters in A431 cells. We discovered striking differences when we examined the kinetics of formation of diacylglycerol (DAG) stimulated by ATP among these cell lines. Thus, ATP stimulated a sustained biphasic increase of DAG in A431 cells, but only a rapid transient increase of DAG formation was observed in 3T3 and 3T6 cells. The breakdown of phosphatidylcholine was stimulated by ATP in A431 cells; however, a significantly reduced effect was displayed in 3T6 cells. Furthermore, we found that the diacylglycerol-kinase inhibitor, 1-monooleoylglycerol, greatly potentiated ATP-stimulated DNA synthesis in A431 cells. Finally, down-regulation of PKC by long-term exposure to phorbol dibutyrate (PDBu) prevented stimulation of DNA synthesis induced by bombesin, vasopressin, or phorbol esters in 3T3 or 3T6 cells, while it had no such effect on ATP-stimulated mitogenesis in the presence of insulin or epidermal growth factor. On the other hand, PDBu-mediated down-regulation of PKC partially inhibited [3H [thymidine incorporation stimulated by ATP in A431 cells. Taken together, we conclude that a protein kinase C-dependent pathway is partially involved in ATP-stimulated DNA synthesis in A431 cells, but a protein kinase C-independent pathway exists in 3T3 and 3T6 cells. Pertussis toxin (PTX) inhibited the sustained phase of DAG formation and the breakdown of phosphatidylcholine stimulated by ATP in A431 cells. This suggests involvement of a PTX-sensitive G protein.     

Guanosine 5'-O-(3-thiotrisphosphate) potentiates both thrombin- and platelet-derived growth factor-induced inositol phosphate release in permeabilized vascular smooth muscle cells. Signaling mechanisms distinguished by sensitivity to pertussis toxin and phorbol esters

We compared the mechanisms by which thrombin and platelet-derived growth factor (PDGF) activate phospholipase C in cultured vascular smooth muscle cells. Thrombin caused a transient (less than 5 min) increase in inositol trisphosphate (IP3) while PDGF caused a sustained (greater than 10 min) increase. Both pertussis toxin and phorbol 12-myristate 13-acetate (PMA) inhibited the thrombin-induced increase in IP3 but neither agent affected the PDGF-induced increase in IP3. To examine the role of GTP binding (G) proteins in the activation of phospholipase C by these two hormones, GTP analogues were introduced into saponin-permeabilized cells. In the absence of hormones, guanosine 5'-O-(3-thiotrisphosphate) (GTP gamma S) caused a progressive increase in IP3 release which was inhibited 55% by PMA (200 ng/ml). In the presence of thrombin, GTP gamma S caused synergistic increase in IP3 release. The synergism between GTP gamma S and thrombin was virtually eliminated by 10 min prior exposure to PMA (200 ng/ml). When PDGF was the hormonal agonist, GTP gamma S also caused synergistic increase in IP3 release and guanosine 5'-O-(2-thiodiphosphate) blunted PDGF-induced IP3 release. However, in contrast to thrombin, the synergism between GTP gamma S and PDGF was unaffected by PMA. Thus, thrombin and PDGF activate phospholipase C by signal transduction systems which differ in kinetic properties and in sensitivity to PMA and pertussis toxin. Despite these differences, both systems appear to involve GTP binding proteins at some step.     

Effects of insulin,pertussis toxin and cholera toxin on protein synthesis and diacylglycerol production in 3T3 fibroblasts: Evidence for a G-protein mediated activation of phospholipase C in the insulin signal mechanism

The rapid increase in protein synthesis that occurs on addition of insulin (1 mU/ml) to stepped-down 3T3 cells was blocked by pre-incubation of the cells with pertussis toxin. Cholera toxin on the other hand stimulated protein synthesis and this effect was insensitive to actinomycin D and inhibited by pro-treatment of the cells with phorbol dibutyrate to deplete cell protein kinase C. Insulin was found to cause a rapid and transient increase in diacylglycerol (DAG) synthesis. The insulin-induced increase in diacylglycerol was blocked by pertussis toxin. Exogenous DAG (10 M) stimulated protein synthesis within 1 hour. The results suggest that insuIin stimulates ribosomal activity through a signal mechanism that involves a G-protein mediated activation of phospholipase C to increase DAG levels.     

GTP gamma S-induced solubilization of actin and myosin from rabbit peritoneal neutrophil membrane

Addition of guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) to the membrane fraction isolated from rabbit peritoneal neutrophils results in the solubilization of several proteins from the membrane. The major proteins are of 180 kDa (myosin) and 43 kDa (actin). The effect is observed with a half-maximum GTP gamma S concentration of 70 microM. The potencies of various nucleotides are compared: GTP gamma S greater than GTP greater than ATP greater than GDP, GMP, cGMP, cAMP. The effect does not require calcium and is not inhibited by using membranes prepared from cells that have been pretreated with pertussis toxin.     

Pertussis toxin-sensitive GTP-binding proteins may regulate phospholipase A2 in response to thrombin in rabbit platelets

Incubation of rabbit platelets with thrombin resulted in rapid accumulations of inositol trisphosphate (IP3) in [3H]inositol-labeled platelets, increases of [3H]arachidonic acid [( 3H]AA) release, and [3H]serotonin secretion from the platelets prelabeled with these labeled compounds. The experiments using phospholipase A2 or C inhibitor suggested that not only phospholipase C but also phospholipase A2 activity plays an important role in serotonin secretion. We then studied the regulatory mechanisms of phospholipase A2 activity. Guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), guanyl-5'-(beta,gamma-iminio)triphosphate), or AlF4- caused a significant liberation of AA in digitonin-permeabilized platelets but not in intact platelets. Thrombin-stimulated AA release was not observed in permeabilized platelets, whereas thrombin acted synergistically with GTP or GTP analogs to stimulate AA release. GTP analog-stimulated AA release was inhibited by guanosine 5'-(2-O-thio)diphosphate) and was also inhibited by decreased Mg2+ concentrations. Thrombin-induced, GTP-dependent AA release, but not IP3 formation, was diminished by 100 ng/ml of pertussis toxin, associated with ADP-ribosylation of membrane 41-kDa protein(s). Thrombin-stimulated AA release from intact platelets and GTP gamma S-stimulated release from permeabilized platelets were both markedly dependent on Ca2+. However, Ca2+ addition could not enhance AA release without GTP gamma S even when Ca2+ was increased up to 10(-4) M in permeabilized platelets. The results show that thrombin-stimulated AA release from rabbit platelets is mainly mediated by phospholipase A2 activity, not by phospholipase C activity, and that Ca2+ is an important factor to the activation of phospholipase A2 but is not the sole factor to the regulation. GTP-binding protein(s) is involved in receptor-mediated activation of phospholipase A2.     

Physiologic activation of protein kinase C limits IL-2 secretion

Interaction of Ag, antibodies against the T cell receptor complex, or mitogenic lectins with T lymphocytes induces hydrolysis of membrane phospholipids leading to the production of diacylglycerol (DAG). DAG then activates the Ca2+- and phospholipid-dependent phosphotransferase, protein kinase C (PKC). Increases in DAG concentrations are transient as is the increase in PKC activity. Phorbol esters, which induce potent, prolonged activation of PKC, augment many T lymphocyte responses, including cell proliferation and secretion of the T cell growth factor IL-2. Therefore, it has been suggested that activation of PKC is a positive regulatory signal in T lymphocytes. We have determined the consequences of transient stimulation of PKC, and of depletion of PKC, on early cell activation signals and on production of IL-2 by the murine lymphoma line LBRM 331A5. When this cell line is depleted of PKC overnight incubation in high concentrations of phorbol esters, lectin-induced IL-2 secretion is augmented. Similarly, mitogen-induced changes in [Ca2+]i and phosphoinositide metabolism were augmented in these cells. In contrast, a short preactivation of PKC abrogated these early transmembrane signaling events. This suggested that normal physiologic activation of PKC may limit cell activation and decrease IL-2 production. We compared the effects of phorbol esters and mezerein, which produce prolonged activation of PKC, with those of diacylglycerol analogs, which induce transient activation of PKC. At concentrations that give similar levels of PKC activation, phorbol esters and mezerein, but not DAG analogs, increased IL-2 secretion. This suggests that prolonged, nonphysiologic activation of PKC is required to augment IL-2 secretion. Therefore, physiologic activation of PKC may not augment T cell activation but instead may function to decrease cell activation and limit IL-2 secretion.     

IL-10 is produced by subsets of human CD4+ T cell clones and peripheral blood T cells.

Murine IL-10 has been reported originally to be produced by the Th2 subset of CD4+ T cell clones. In this study, we demonstrate that human IL-10 is produced by Th0, Th1-, and Th2-like CD4+ T cell clones after both Ag-specific and polyclonal activation. In purified peripheral blood T cells, low, but significant, levels of IL-10 were found to be produced by the CD4+CD45RA+ population, whereas CD4+CD45RA- "memory" cells secreted 5- to 20-fold higher levels of IL-10. In addition, IL-10 was produced by activated CD8+ peripheral blood T cells. Optimal induction of IL-10 was observed after activation by specific Ag and by the combination of anti-CD3 mAb and the phorbol ester tetradecanoyl phorbol acetate, whereas the combination of calcium ionophore A23187 and 12-O-tetradecanoylphorbol-13-acetate acetate was a poor inducer of IL-10 production. Kinetic studies indicated that IL-10 was produced relatively late as compared with other cytokines. Maximal IL-10 mRNA expression in CD4+ T cell clones and purified peripheral blood T cells was obtained after 24 h, whereas maximal IL-10 protein synthesis occurred between 24 h and 48 h after activation. No differences were observed in the kinetics of IL-10 production among Th0, Th1-, and Th2-like subsets of CD4+ T cell clones. The results indicate a regulatory role for IL-10 in later phases of the immune response.     

Histamine acts directly on human T cells to inhibit interleukin-2 and interferon-gamma production

Histamine acts directly on human T cells to inhibit lymphokine production without the involvement of accessory cells. Histamine inhibits the production of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) by purified human peripheral T cells activated in the presence of either intact monocytes or metabolically inactive fixed Raji and U698 cells as accessory cells. Purified T cells do not respond more than marginally to staphylococcal enterotoxin A (SEA) or phytohemagglutinin (PHA) in the absence of accessory cells. However, activation by the phorbol ester PMA in conjunction with either PHA or the calcium ionophore A23187 induces large amounts of IFN-gamma and IL-2. Histamine suppresses the lymphokine production in these pure T-cell cultures to a similar extent as in monocyte-containing cultures. Histamine is also shown to suppress DNA synthesis by purified T cells cultivated at a low cell density, eliminating any possible involvement of small numbers of contaminating accessory cells. In vitro preactivated T cells are shown to retain their capacity to respond to histamine when stimulated by PMA and A23187 or by mitogen in the presence of Raji cells. The conclusion that histamine acts directly on T cells and does not require accessory cells to induce suppression is further confirmed by the demonstration that IL-2 production by the human T-cell leukemia line Jurkat was significantly suppressed by histamine in a H-2 receptor-restricted manner.     

Signal transduction in cells following binding of chemoattractants to membrane receptors

Binding of chemoattractants to specific cell surface receptors on human polymorphonuclear leukocytes (PMNs) initiates a variety of biologic responses, including directed migration (chemotaxis), release of superoxide anions, and lysosomal enzyme secretion. Chemoattractant receptors belong to a large class of receptors which utilize the hydrolysis of polyphosphoinositides to initiate Ca2+ mobilization and cellular activation. Receptor occupancy leads to phospholipase C-mediated hydrolysis of polyphosphoinositol 4,5-bisphosphate (PIP2) yielding inositol 1,4,5-trisphosphate (IP3) and 1,2 sn-diacylglycerol (DAG). These products synergize to initiate cell activation via calcium mobilization (IP3) and protein kinase C activation (DAG). Pertussis toxin, which ADP-ribosylates and inactivates some GTP binding proteins (G proteins), abolishes all chemoattractant-induced responses, including Ca2+ mobilization, IP3 and DAG production, enzyme secretion, superoxide production and chemotaxis. Direct evidence for chemoattractant receptor: G protein coupling was obtained using PMN membrane preparations which contain a Ca2+-sensitive phospholipase C. Hydrolysis of polyphosphoinositides at resting intracellular Ca2+ levels (100 nm) was only observed when the membranes were stimulated with the chemoattractant N-formyl-methyl-leucyl-phenylalanine (fMet-Leu-Phe) in the presence of GTP. Myeloid cells contain two distinct pertussis toxin substrates of similar molecular weight (40 and 41 kD). The 41 kD substrate resembles Gi, whereas a 40 kD substrate is physically associated with a partially purified fMet-Leu-Phe receptor preparation and may therefore represent a novel G protein involved in chemoattractant-stimulated responses. Metabolism of 1,4,5-IP3 to inositol proceeds via two distinct pathways in PMNs: (1) degradation to 1,4-IP2 and 4-IP1 or (2) conversion to 1,3,4,5-IP4, 1,3,4-IP3, 3,4-IP2 and 3-IP1. Initial formation (0-30 s) of 1,4,5-IP3 and DAG occurs at ambient intracellular Ca2+ levels, whereas formation of 1,3,4-IP3 and a second sustained phase of DAG production (30 s-10 min) require elevated cytosolic Ca2+ influx. The later peak of DAG, which is not derived from phosphoinositides, appears to be required for stimulation of respiratory burst activity. Products formed during activation can feed back to attenuate chemoattractant receptor-mediated stimulation of phospholipase C by uncoupling receptor-G protein-phospholipase C interaction.     

Trypanosoma cruzi: infection of cultured human endothelial cells alters inositol phosphate synthesis

Infection of cultured endothelial cells with Trypanosoma cruzi alters intracellular Ca2+ homeostasis. To help understand the biochemical basis for this phenomenon, we determined the influence of infection on inositol phosphate formation in a broken cell preparation. Inositol phosphates participate in the regulation of cytosolic Ca2+. In uninfected endothelial cells, bradykinin guanosine 5'-O-thiophosphate (GTP tau S), and calcium all stimulated inositol phosphate (IP1), inositol bisphosphate (IP2), and inositol trisphosphate (IP3) formation within 5 sec of incubation. At longer periods of incubation with GTP tau S and bradykinin, formation of IP1 was linear for 30 sec, whereas the rate of IP2 and IP3 generation was maximal at 20 and 5 sec, respectively. Second, infection markedly changed these aspects of inositol phosphate generation. First, unstimulated (basal) levels of IP1 and IP3 were markedly increased over those levels in membranes of uninfected cells. Infection decreased the rate of formation for the three inositol phosphates in response to GTP tau S and bradykinin. Finally, infection diminished the magnitude of inositol phosphate synthesis in response to Ca2+ for IP1, IP2, and IP3, respectively. Studies on G proteins using cholera and pertussis toxin were carried out to determine if the infection-associated changes in inositol phosphate generation could be attributed to functional changes in these regulatory proteins known to participate in the activation of phospholipase C. Infection markedly decreased the magnitude of cholera and pertussis toxin-dependent ADP ribosylation, as compared to control uninfected cells. Incubation of uninfected endothelial cells with cholera and pertussis toxin also decreased the magnitude of cholera and pertussis toxin ADP ribosylation. Despite the similar effects of infection and toxin treatment on subsequent toxin-catalyzed ADP ribosylation, toxin treatment did not influence inositol phosphate generation. Collectively, these results demonstrate an influence of infection on receptor-dependent and -independent synthesis of inositol phosphates, possibly by an action on phospholipase C. The results help to explain the apparent infection-associated increase in basal Ca2+ previously observed and suggest that interference with signal transduction may be a consequence of the presence of the parasite.     

Induction of the fibrinogen receptor on human platelets by intracellular mediators

We have used platelets permeabilized with saponin to examine the mechanism by which platelet activation causes the exposure of surface receptors for fibrinogen. Receptor exposure was detected using 125I-fibrinogen and 125I-PAC1, a monoclonal antibody specific for the activated form of the fibrinogen receptor. The potential mediators that were studied included guanyl-5'-yl imidodiphosphate (Gpp(NH)p) and guanosine 5'O-(thiotriphosphate) (GTP gamma S), which cause G protein-dependent phospholipase C activation in platelets; inositol 1,4,5-triphosphate (IP3), which causes Ca2+ release from the platelet dense tubular system; and diacylglycerol and phorbol ester, which activate protein kinase C. Each of these molecules caused fibrinogen and PAC1 binding. The effect of IP3 was mimicked by raising the cytosolic free Ca2+ concentration in the permeabilized platelets. However, IP3 and Ca2+-induced PAC1 binding were abolished by indomethacin or aspirin, which had no effect on PAC1 binding caused by Gpp(NH)p, phorbol ester, or diacylglycerol. This suggests that the response to IP3 and Ca2+ is due to the formation of metabolites of arachidonic acid. One such metabolite, TxA2, is believed to activate platelets by stimulating G protein-dependent phosphoinositide hydrolysis. Indeed, we found that the G protein inhibitor guanyl-5'-yl thiophosphate (GDP beta S) inhibited PAC1 binding caused by a thromboxane A2 analog (U46619), IP3, and Ca2+, but had no effect on diacylglycerol or phorbol ester-induced PAC1 binding. Thrombin-induced PAC1 binding and phosphoinositide hydrolysis were also inhibited by GDP beta S and by pertussis toxin. Increasing the thrombin concentration overcame the inhibition of PAC1 binding caused by GDP beta S but did not overcome the inhibition of phosphoinositide hydrolysis. These observations demonstrate that fibrinogen receptor exposure occurs by at least two routes. One of these, in response to agonists such as thrombin and U46619, is initiated by G protein-dependent phosphoinositide hydrolysis and involves the formation of IP3 and diacylglycerol. IP3 appears to act by stimulating Ca2+-dependent arachidonic acid metabolism which, in turn, triggers further phosphoinositide hydrolysis. Diacylglycerol acts by stimulating protein kinase C. A second route is activated by high concentrations of thrombin and is independent of phosphoinositide hydrolysis.