Transmembrane signaling during natural killer cell-mediated cytotoxicity. Regulation by protein kinase C activation

NK cells can mediate either FcR-dependent cytotoxicity against antibody-coated target cells or direct cytotoxicity against a variety of tumor cells. We used homogeneous, cloned populations of CD16+/CD3- human NK cells to characterize and compare the transmembrane signaling mechanisms used during these alternative forms of cytotoxicity. Cross-linkage of NK cell FcR with anti-FcR (anti-CD16) mAb or direct binding to NK-sensitive tumor targets resulted in a rapid release of inositol phosphates and increases in [Ca2+]i. The receptor-dependent [Ca2+]i increase (as monitored in indo-1 loaded NK cells by flow cytometry) consisted of an initial release of calcium from intracellular stores, followed by a sustained influx of calcium across the plasma membrane. To assess the potential regulatory feedback role of protein kinase C (PKC) activation in these proximal signaling events, NK cells were pretreated with either PKC-activating phorbol esters, nonactivating phorbol ester homologs, or synthetic diacylglycerols. Brief pretreatment with activating phorbol esters rapidly inhibited, in a concentration-dependent manner, both phosphoinositide hydrolysis and increases in [Ca2+]i induced by FcR ligation, whereas pretreatment with an inactive phorbol ester had no effect. This acute inhibitory effect was not explained by FcR down-regulation, which occurred with more prolonged exposure to phorbol esters. In contrast, the phosphoinositide turnover and [Ca2+]i increase in NK cells stimulated with NK-sensitive tumor targets were not affected by prior exposure to PKC-activating phorbol esters. This differential regulatory effect of phorbol ester on proximal signaling was paralleled by a corresponding effect on cytotoxicity, i.e., phorbol ester-induced activation of PKC inhibited FcR-dependent cytotoxicity, but did not alter direct cytotoxicity against NK-sensitive tumor cells. These results indicate that PKC activation can differentially regulate alternative forms of NK cell-mediated cytotoxicity by rapidly and specifically desensitizing the FcR.     

NKR-P1, an activating molecule on rat natural killer cells, stimulates phosphoinositide turnover and a rise in intracellular calcium

NKR-P1 is a 60-kDa homodimer expressed on all rat NK cells. Previous studies by others suggest that NKR-P1 may play a role in NK cell activation because antibody to NKR-P1 stimulates the release of granules from NK cells, and anti-NKR-P1 causes redirected lysis by activated NK cells against targets that express FcR. To examine the mechanism of transmembrane signaling by NKR-P1, we studied the rat NK cell line, RNK-16. We here demonstrate that F(ab')2 antibody to NKR-P1 stimulates phosphoinositide turnover and a rise in intracellular calcium within RNK-16 cells. The response is augmented by cross-linking the F(ab')2 antibody. The phosphoinositide/calcium pathway is also stimulated by NKR-P1 in activated rat NK cells, although no response is detectable in polymorphonuclear cells, which also express NKR-P1. We also demonstrate that RNK-16 cells kill the anti-NKR-P1 (3.2.3) hybridoma and that exposure to the hybridoma target cells stimulates phosphoinositide turnover in RNK-16 cells. Both killing and phosphoinositide turnover are inhibited by F(ab')2 anti-NKR-P1, implicating NKR-P1 in both responses. In contrast, neither cytotoxicity nor phosphoinositide turnover is appreciably blocked by F(ab')2 anti-NKR-P1 in response to YAC-1 targets. Thus, with either target, killing is linked to phosphoinositide turnover, but killing of YAC-1 involves pathways that differ from those that direct killing of the anti-NKR-P1 hybridoma. Our studies support the hypothesis that NKR-P1 may serve as an activating cell-surface receptor on NK cells, and they clarify the mechanisms by which it activates NK cells.     

Prostaglandin E receptor enhancement of catecholamine release may be mediated by phosphoinositide metabolism in bovine adrenal chromaffin cells

In the presence of ouabain, prostaglandin (PG) E2 stimulated a gradual secretion of catecholamines from cultured bovine adrenal chromaffin cells. PGE2 or ouabain alone evoked a marginal secretory response. The synergism of ouabain was also observed with muscarine. PGE2, like muscarine, induced a concentration-dependent formation of inositol phosphates: rapid rises in inositol trisphosphate and inositol bisphosphate followed by a slower accumulation of inositol monophosphate. This effect on phosphoinositide metabolism was accompanied by an increase in cytosolic free Ca2+. The potency of PGs (PGE2 greater than PGF2 alpha greater than PGD2) to stimulate catecholamine release was well correlated with that to affect phosphoinositide metabolism and that to increase the level of intracellular Ca2+. PGE2 did not stimulate cAMP generation significantly in bovine chromaffin cells. The effect of PGE2 on catecholamine release was mimicked by 12-O-tetradecanoylphorbol 13-acetate and A23187, but not by the cAMP analogue dibutyryl cAMP nor by forskolin. These results indicate that PGE2 may enhance catecholamine release from chromaffin cells by activating protein kinase C in concert with the increment of intracellular Ca2+.     

Alternative mechanisms of natural killer cell activation during herpes simplex virus infection

Although NK cells can kill both malignant cells and virus-infected cells without prior sensitization, it has remained unclear whether the mechanism by which an NK cell is activated in the presence of a tumor cell is similar to that induced by the presence of a virus-infected cell. In our experimental system using homogeneous populations of cloned human CD16+ NK cells, we found that HSV-infected target cells do not induce in the NK cells the same pharmacologically-active second messengers elicited by NK-sensitive tumor cells. Although phosphoinositide turnover and calcium signaling were generated in NK cells exposed to NK-sensitive tumor cells, the recognition of HSV-infected cells by NK cells did not result in similar transmembrane signaling. Furthermore, depending on the cell type infected by HSV, alternative mechanisms of cytotoxicity were employed. HSV-infected foreskin fibroblasts were rapidly and selectively killed by cloned NK cells without a requirement for IFN or accessory cells. In contrast to this direct cytotoxicity against HSV-infected foreskin fibroblasts, NK cell-mediated cytotoxicity against an HSV-infected fibrosarcoma cell line (1591) was dependent on IFN-alpha production by accessory cells. Importantly, in both systems of cytotoxicity, IFN-alpha activation of NK cells resulted in augmented killing against both infected and uninfected targets. These results suggest that NK cell activation induced during antiviral immunity is distinct from activation elicited during an antitumor response. These differences include the utilization of alternative forms of signal transduction and alternative mechanisms of cytotoxicity.     

Synthetic diacylglycerols trigger an increase of intracellular free calcium in promyelocytic HL60 cells

Phosphoinositide turnover is known to play an important role in intracellular free calcium homeostasis through the inositol trisphophate-mediated release of calcium from intracellular stores. We find that the other product of phosphoinositide turnover, 1,2-diacylglycerol, elicits an increase in intracellular free calcium in HL60 cells which is due, at least in part, to release of calcium from intracellular stores. This effect is specific for calcium, since intracellular sodium and potassium levels and cellular volume were unaffected. Concomitant with the intracellular calcium increase, we find an increase in cellular inositol trisphosphate levels, suggesting that the effect of diacylglycerol on calcium may be mediated by inositol trisphosphate. Diacylglycerols also stimulate calcium efflux. This stimulation is not simply due to the increase in intracellular calcium. These effects appear not to be mediated through stimulation of a phorbol ester-activatable protein kinase C (Ca2+/phospholipid-dependent enzyme) since phorbol esters do not elicit an increase in cytoplasmic free calcium or an increase in calcium efflux.     

Muscarinic release of inositol trisphosphate without mobilization of calcium in bovine adrenal chromaffin cells

Chromaffin cells of bovine adrenal medulla release catecholamines in response to activation of nicotinic ACh receptors which open voltage-sensitive calcium channels. Catecholamine secretion by exocytosis requires an increase in cytosolic free calcium. The cells also possess muscarinic ACh receptors but muscarinic agents do not provoke catecholamine release. Quin-2 studies show that they do not increase cytosolic free Ca2+ concentration, but unlike the nicotinic agents, they cause phosphoinositide hydrolysis. Muscarinic stimulation leads to rapid loss of labelled phosphatidylinositol 4-phosphate and of phosphatidylinositol 4,5-bisphosphate. At the same time there is release of inositol trisphosphate, inositol bisphosphate and inositol phosphate. In a number of other cells inositol trisphosphate may act as a second messenger releasing Ca2+ from storage sites in the endoplasmic reticulum but this is not its function in bovine chromaffin cells.     

Monoclonal anti-CD23 antibodies induce a rise in [Ca2+]i and polyphosphoinositide hydrolysis in human activated B cells. Involvement of a Gp protein

Transduction through the CD23 molecule (Fc epsilon RII) was analyzed in human activated B lymphocytes using anti-CD23 mAb. B cell blasts expressing an increased amount of surface CD23 molecule were obtained by stimulation of normal peripheral blood B lymphocytes with Staphylococcus aureus strain Cowan I and IL-4. Anti-CD23 mAb were found to trigger polyphosphoinositide hydrolysis in these cells (and also in tumoral B cells expressing spontaneously CD23) and a rise in [Ca2+]i which could be attributed to mobilization from cytoplasmic pools. This increase in [Ca2+]i could be mimicked, with a comparable time-course, by the addition of InsP3 to permeabilized B cell blasts indicating that the increase in inositol phosphate accumulation induced by the antibodies was due to a preferential attack of phosphatidylinositol-bisphosphate by a specific phosphoinositidase C (PIC). In permeabilized cells, raising the free calcium concentration above 3 microM was found to induce polyphosphoinositides hydrolysis and to activate directly the PIC. Addition of 100 microM GTP-tetralithium salt, a non-hydrolyzable analogue of GTP, also resulted in an increased accumulation of inositol phosphates. A Ca2(+)-dependent PIC, linked to a GTP-binding protein (Gp protein), can thus be activated in B cell blasts. Addition of anti-CD23 antibodies to permeabilized B cells in the presence of a physiologic concentration of Ca2+ (100 nM) evoked, within 10 min, a rise in the various inositol phosphates. This ability of anti-CD23 antibodies to activate PIC was enhanced in the presence of GTP-tetralithium salt 100 microM. By contrast, preincubation with GDP-trilithium salt, a nonhydrolyzable analogue of GDP, caused a marked reduction in the release of inositol phosphates. Preincubation of B cell blasts with Pertussis toxin resulted in a total inhibition of the capacity of the toxin to ADP-ribosylate a 41-kDa protein, probably of the Gi type; in these conditions, no modification of anti-CD23-elicited polyphosphoinositide hydrolysis could be detected. These results suggest that the CD23 molecule may be coupled to the phosphoinositide signaling pathway by a GTP-dependent component that is insensitive to Pertussis toxin.     

Cyclic AMP- and inositol phosphate-independent inhibition of Ca2+ influx by cholera toxin in CD3-stimulated Jurkat T cells. A study with a cholera toxin-resistant cell variant and the Ca2+ endoplasmic reticulum-ATPase inhibitor thapsigargin.

In this report, we describe a Jurkat cell variant, termed JCT8, the selection of which is based upon its resistance to cell-growth inhibition mediated by the holotoxin of Vibrio cholerae, cholera toxin (CT). JCT8 cells exhibit normal cAMP production in response to various cAMP inducers, including CT, together with conserved ADP ribosylation in vitro of G-protein Gs alpha by the A subunit of the toxin. However, after a 4-h pretreatment with CT, JCT8 cells have a conserved expression of cell-surface CD3 molecules. These effects are in contrast to those elicited by the toxin in long term PGE2-desensitized Jurkat cells, which remain as sensitive as the wild type to the inhibitory action of CT on cell growth and CD3 cell-surface expression, despite poor responsiveness to CT with regard to cAMP production. In JCT8 cells, Ca2+ mobilization induced via the CD3/TCR is maintained after CT treatment contrasting with its complete suppression in the wild-type and in the PGE2-desensitized cells. However, as in the other cell types, CT still suppresses Ca2+ influx in JCT8 cells. Increase in inositol phosphates by CD3 stimulation of JCT8 cells, including of inositol 1,4,5-triphosphate (I(1,4,5)P3), is only partially antagonized by CT. This suggests either that in JCT8 cells there is a different susceptibility of Ca2+ mobilization and influx to partial inhibition by CT of CD3-triggered phospholipase C (PLC)-induced phosphoinositide hydrolysis or that an additional and PLC-independent suppressive effect of the toxin on Ca2+ influx may exist. To investigate this particular point further, we use Thapsigargin, a Ca(2+)-endoplasmic reticulum ATPase inhibitor that can mobilize in human T lymphocytes I(1,4,5)P3-dependent intracellular Ca2+ pools by a PLC-independent pathway. We demonstrate that the Ca2+ influx triggered in the wild-type Jurkat cells or in JCT8 cells by Thapsigargin is antagonized by CT. The present data are therefore consistent with the idea that CT specifically impairs in the Jurkat T cell model the entry of Ca2+ from extracellular spaces by a mechanism independent not only from cAMP but also in part from inhibition by the toxin of phosphoinositide hydrolysis.     

Lipid signaling in T-cell development and function

Second messenger molecules relay, amplify, and diversify cell surface receptor signals. Two important examples are phosphorylated D-myo-inositol derivatives, such as phosphoinositide lipids within cellular membranes, and soluble inositol phosphates. Here, we review how phosphoinositide metabolism generates multiple second messengers with important roles in T-cell development and function. They include soluble inositol(1,4,5)trisphosphate, long known for its Ca(2+)-mobilizing function, and phosphatidylinositol(3,4,5)trisphosphate, whose generation by phosphoinositide 3-kinase and turnover by the phosphatases PTEN and SHIP control a key "hub" of TCR signaling. More recent studies unveiled important second messenger functions for diacylglycerol, phosphatidic acid, and soluble inositol(1,3,4,5)tetrakisphosphate (IP(4)) in immune cells. Inositol(1,3,4,5)tetrakisphosphate acts as a soluble phosphatidylinositol(3,4,5)trisphosphate analog to control protein membrane recruitment. We propose that phosphoinositide lipids and soluble inositol phosphates (IPs) can act as complementary partners whose interplay could have broadly important roles in cellular signaling.     

Cyclic AMP concentrations modulate both calcium flux and hydrolysis of phosphatidylinositol phosphates in mouse T lymphocytes

Activation of T cells by lectins or mAb directed at components of the Ag-specific TCR results in hydrolysis of phosphorylated derivatives of phosphatidylinositol and an increase in intracellular free calcium concentration (Cai). We report that cholera toxin, which activates adenylate cyclase by ADP ribosylation of a G protein, also reduces both inositol phosphate (IP) production and the rise in Cai in Con A-stimulated murine T cells. We find that similar dose-dependent inhibitory effects can be induced by each of four other agents that raise cAMP levels in such cells: forskolin, PGE2, 2-chloroadenosine, and isoproterenol. The effects of these agents on IP production are reversible and therefore do not simply reflect cytotoxicity. Activation by PHA and by antibody to the T3-epsilon-chain of the TCR complex are also inhibited by agents that increase intracellular cAMP. Thus, changes in cAMP concentration seem to regulate both IP production and the Ca2+ response, two early components of the mitogen-induced activation process.     

Mechanisms of action of calcium-mobilizing agonists: some variations on a young theme

It is now accepted that many hormones and neurotransmitters exert their effects through G protein-mediated activation of a phospholipase C, which breaks down phosphatidylinositol bisphosphate. This releases inositol trisphosphate, which mobilizes intracellular calcium, and diacylglycerol, which, in turn, activates protein kinase C. However, recent evidence indicates that other mechanisms are involved. In some cells, the increases in cytosolic calcium elicited within 1-2 s by high concentrations of agonists or at later times by low, physiological concentrations of agonists occur without any detectable changes in inositol phosphates and calcium mobilization, and result from the opening of plasma membrane channels that are permeable to Ca2+. This response appears to be mediated more directly by G proteins. These findings question the postulated roles of inositol phosphates and calcium mobilization in the stimulation of calcium influx. Measurements of the mass and fatty acid composition of the inositol phospholipids and of the diacylglycerol and phosphatidic acid generated by agonists in several cell types indicate that phosphatidylinositol bisphosphate is probably a minor source of these lipids. On the other hand, measurements of phosphatidylcholine, choline, and phosphocholine indicate that this phospholipid is a major source, and that its breakdown involves both phospholipase C and D. These findings indicate that phosphatidylcholine breakdown may be more important than phosphoinositide hydrolysis in the regulation of protein kinase C and perhaps other cell functions.     

Role of the CD45 (T-200) molecule in anti-CD3-triggered T cell-mediated cytotoxicity

NK-depleted human peripheral blood lymphocytes can be modulated with anti-CD3 to kill certain targets during 3-hr cytotoxicity assays. When triggered by anti-CD3 antibody, these effector T cells killed only NK-sensitive targets, such as K562 and HEL 92.1.7, and NK-resistant targets, such as Daudi, whose killing is inhibited by anti-CD45 (T-200) monoclonal antibodies, such as 13.3. NK-sensitive targets, MOLT-4, U266/AF10, Jurkat, and CCFR-CEM, and 10 NK-resistant cell lines, including Raji, IM-9, U698, U937, and GM-1056, whose killing is not inhibited by anti-CD45 monoclonal antibodies, were not killed by alpha-CD3-T effectors, suggesting that the CD45 molecule may be involved in the killing process. Anti-CD3-triggered T cell killing of target cells was inhibited greater than 95% by the monoclonal antibody 13.3. This inhibition of cytotoxicity by 13.3 was not due to competition of this IgG1 antibody for Fc receptor binding site on the target cell, since the IgG1 monoclonal antibody anti-beta 2-microglobulin did not block cytotoxicity. Single cell assays and calcium pulse assays showed that CD45 is involved in a postbinding, pre-calcium-dependent stage, similar to that shown for NK cytotoxicity. There was a relative shift of importance of different epitopes of CD45 in anti-CD3-T cytotoxicity compared to NK cytotoxicity. Anti-CD45 antibodies which bind to the C terminus end of the molecule played a more important role in anti-CD3-T cytotoxicity than NK cytotoxicity. Thus, a subset of T cells exists that exhibits anti-CD3-triggered non-MHC-restricted killing of certain NK-sensitive and NK-resistant targets in association with a CD45 molecule which is functionally different from the NK CD45 molecule.     

Phosphoinositide metabolism and the calcium response to concanavalin A in S49 T-lymphoma cells. A comparison with thymocytes.

Comparisons were made between transformed S49 T-lymphoma cells and normal murine thymocytes in their polyphosphoinositides, inositol polyphosphates and cytosolic free calcium concentrations ([Ca2+]i), and the effects of the T-cell mitogen concanavalin A (Con A) on these properties. 1. The ratios of the polyphosphoinositides to phosphatidylinositol in both exponential-phase S49 cells and mitogen-stimulated thymocytes (G1 phase) were greater than in quiescent (G0-phase) thymocytes. 2. In response to Con A, the amount of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) in S49 cells decreased slightly (17% in 30 min), and this was sufficient to account for the small amounts of inositol phosphates that accumulated. In contrast, it has been shown previously that Con A stimulates a rapid resynthesis of PtdInsP2 in thymocytes and the amounts of inositol phosphates released rapidly exceed the steady-state amount of the PtdInsP2 precursor [Taylor, Metcalfe, Hesketh, Smith & Moore (1984) Nature (London) 312, 462-465]. 3. The [Ca2+]i did not differ significantly in S49 cells and thymocytes before the addition of Con A, and the increases in [Ca2+]i in response to Con A were similar in both types of cell. 4. The [Ca2+]i increase in response to Con A was inhibited by similar concentrations of intracellular cyclic AMP (2-10 microM) in S49 cells and thymocytes, suggesting that similar regulatory mechanisms act on this response in both types of cell. The data demonstrate that the basal [Ca2+]i and phosphoinositide metabolism is similar in both the normal cells and their transformed counterparts. In addition, they suggest that the activated Con A receptors generate very similar signals in the two cell types, and that any perturbations of primary signal transduction to the secondary phosphoinositide and [Ca2+]i responses in the S49 phenotype are quantitative rather than qualitative.     

An immobilized metal ion affinity adsorption and scintillation proximity assay for receptor-stimulated phosphoinositide hydrolysis

A novel approach to measuring receptor-stimulated phosphoinositide hydrolysis was developed based on the principles of immobilized metal ion affinity chromatography (IMAC) and scintillation proximity assay (SPA). Hard Lewis metal ions, such as Zr(4+), Ga(3+), Al(3+), Fe(3+), Lu(3+), and Sc(3+), were immobilized on SPA beads via metal chelate and utilized as affinity ligands to entrap inositol phosphates. [3H]Inositol phosphates bound to IMAC-SPA beads through the strong interaction of their phosphate group with the immobilized metal ions. The binding brought [3H]inositol phosphates in close proximity to the scintillant embedded in the SPA beads, thereby allowing the radioactivity to be quantified. Quantification of [3H]inositol phosphate production in cells preincubated with [3H]inositol provided a highly sensitive measurement of phosphoinositide hydrolysis. The utility of this approach was demonstrated in measuring the response mediated by the G-protein-coupled neurokinin NK1 receptor and the tyrosine kinase-linked platelet-derived growth factor (PDGF) receptor. Substance P stimulated phosphoinositide hydrolysis concentration-dependently in CHO cells expressing NK1 receptors with a maximal 12-fold increase in inositol phosphate production. Similarly, PDGF-BB stimulated a 5-fold increase in phosphoinositide hydrolysis in quiescent Swiss 3T3 cells. This new approach is highly sensitive, fast, simple, easily performed on 96-well plates, and amenable for high-throughput screening.     

Effects of diacylglycerol and inositol trisphosphate on steroidogenesis by ovarian granulosa from pigs

In addition to increasing cyclic adenosine 3',5'-monophosphate (cAMP) levels, luteinizing hormone (LH) stimulation of granulosa results in phosphoinositide hydrolysis producing inositol trisphosphate (IP3) and diacylglycerol. The roles of these putative second messengers were investigated by measuring production of progesterone and inositol phosphates by granulosa from medium-sized porcine follicles (3-7 mm) after 15 min incubation with or without LH (1 microgram/ml), 5 microM dibutyryl cAMP (dbcAMP), or 5 microM 1-oleoyl,2-acetylglycerol (OAG). Compared to a control rate of 5.4 pmoles/10(7) cells/15 min, LH and dbcAMP stimulated progesterone production to 12.8 and 15.9 pmoles, respectively, and OAG decreased progesterone production to 3.7 pmoles. LH also stimulated inositol phosphate (IP) and bisphosphate (IP2) accumulations by approximately 5-fold and IP3 accumulation by 20-fold. In experiments where granulosa were premeabilized with saponin, LH, dbcAMP, and IP3 stimulated progesterone production from 1.3 pmol in control cells to 5.2, 3.2, and 5.1 pmol, respectively, and OAG decreased progesterone production to 1.0 pmol. LH stimulated accumulation of all inositol phosphates in permeabilized cells, whereas the addition of IP3 only increased IP2 and IP3 accumulations. In granulosa preincubated with 0.9 mM [ethylenebis(oxyethylenenitrilo)] tetraacetic acid, A23187 increased progesterone production from 3.7 to 5.8 pmol. Addition of 1-20 nmoles IP3 to 10(7) granulosa incubated in a Ca2+-free medium increased Ca2+ efflux linearly. These data suggest that IP3 may have a role in regulating steroid production in granulosa by regulating intracellular Ca2+.     

Studies on molecular regulation of phagocytosis in neutrophils. Con A-mediated ingestion and associated respiratory burst independent of phosphoinositide turnover, rise in [Ca2+]i, and arachidonic acid release

The role of the activation of phosphoinositide turnover and of the increase in cytosolic free calcium, [Ca2+]i, in the phagocytosis and associated activation of the respiratory burst was investigated. We report the results obtained on the phagocytosis of yeast cells mediated by Con A in normal and in Ca2+-depleted human neutrophils. In normal neutrophils the phagocytosis was associated with a respiratory burst, a stimulation in the formation of [3H] inositol phosphates and [32P]phosphatidic acid, the release of [3H]arachidonic acid, and a rise in [Ca2+]i. Ca2+-depleted neutrophils are able to perform the phagocytosis of yeast cells mediated by Con A and to activate the respiratory burst without stimulation of [3H]inositol phosphates and [32P]phosphatidic acid formation, [3H]arachidonic acid release, and rise in [Ca2+]i. In both normal and Ca2+-depleted neutrophils the phagocytosis and the associated respiratory burst, 1) were inhibited by cytochalasin B; 2) were insensitive to H-7, an inhibitor of protein kinase C; and 3) did not involve GTP-binding protein sensitive to pertussis toxin. These findings indicate that the activation of phosphoinositide turnover, the liberation of arachidonic acid, the rise in [Ca2+]i, and the activity of protein kinase C are not necessarily required for ingestion of Con A-opsonized particles and for associated activation of the NADPH oxidase, the enzyme responsible for the respiratory burst. The molecular mechanisms of these phosphoinositide and Ca2+-independent responses are discussed.     

Inositol trisphosphate independent increase of intracellular free calcium and amylase secretion in pancreatic acini

It is generally believed that the activation of various cell surface receptors results in the phospholipase C-catalyzed production of inositol trisphosphate which, in turn, increases the intracellular concentration of free Ca2+ by stimulating its release from nonmitochondrial sources. We have investigated both the production of inositol trisphosphate and changes in intracellular Ca2+ concentration in rat pancreatic acini in response to caerulein and CCK-JMV-180, two analogs of cholecystokinin. Both of these analogs cause comparable increases in the rate of amylase secretion and in intracellular Ca2+ concentration but their effects on inositol phosphate generation are dramatically different; caerulein stimulates significant production of inositol phosphates within 1 min of its addition, whereas no detectable levels of inositol phosphates were generated within the same time after addition of CCK-JMV-180. These results suggest that the CCK-JMV-180 stimulated release of intracellular Ca2+ is not mediated by inositol trisphosphate but some other as yet unidentified messenger.     

Mutational analysis of antigen receptor regulation of B lymphocyte growth. Evidence for involvement of the phosphoinositide signaling pathway.

Stimulation of the antigen receptor of WEHI-231 B lymphoma cells with anti-receptor antibodies (anti-IgM) induces irreversible growth arrest. Anti-IgM stimulates two kinds of transmembrane signaling events, phosphorylation of proteins on tyrosyl residues and breakdown of inositol phospholipids, which results in increases of inositol phosphates, diacylglycerol, and calcium. The roles of these reactions in mediating the growth arrest of the B lymphoma cells have not been established. To examine this issue, we took a genetic approach. Mutants of WEHI-231 cells were isolated that were resistant to anti-IgM-induced growth arrest. Five out of seven independent mutants analyzed had normal cell-surface expression of antigen receptors. Although each of these five mutants had tyrosine protein phosphorylation patterns comparable to wild-type cells, they exhibited alterations in the phosphoinositide signaling pathway. Four of the mutants had decreased phosphoinositide breakdown, probably due to an alteration in phospholipase C. Decreased second messenger production may be responsible for the growth-resistant phenotype. Full growth arrest was restored upon addition of the calcium ionophore ionomycin, suggesting that the limiting second messenger was intracellular free calcium. The final mutant appeared to be altered in a component(s) that responds to diacylglycerol and calcium. Taken together, these results provide further evidence that the phosphoinositide pathway is at least partly responsible for mediating antigen receptor regulation of B lymphoma cell growth.     

ATP stimulates inositol phosphates accumulation and calcium mobilization in a primary culture of rat aortic myocytes

The effects of extracellular ATP on phosphoinositide metabolism and intracellular Ca2+ concentration were studied in a primary culture of rat aortic myocytes. ATP increases the level of inositol phosphates, the putative second messenger for Ca2+ mobilization. No saturation of inositol phosphates accumulation is obtained (up to 10(-2) M ATP). Under the same conditions, ATP rapidly mobilizes intracellular Ca2+ in fura-2 loaded myocytes. The mobilization of intracellular Ca2+ is dose-dependent (maximal at 10(-4) M ATP), and is not affected by addition of EGTA. It is concluded that the receptors mediating the cytosolic increase of Ca2+ are of the P2-purinoceptor subtype. The physiological functions of these receptors are not presently known.     

Activation of phospholipase D: a signaling system set in motion by perturbation of the T lymphocyte antigen receptor/CD3 complex.

A number of cellular signaling systems are called into play by interaction of the T lymphocyte antigen receptor/CD3 complex with its cognate antigen. Well-described signaling systems include phosphoinositide turnover, tyrosine phosphorylation, protein kinase C activation, and increased cytosolic calcium. We have explored the possibility that another recently described signaling system, activation of phospholipase D, may be operative. Data presented here demonstrate that stimulation of Jurkat T cells with anti-CD3 antibodies or phorbol esters resulted in activation of phospholipase D, as measured by production of phosphatidylethanol and phosphatidic acid. The combination of anti-CD3 antibody plus phorbol ester led to a greater than additive production of phosphatidylethanol and to the additive production of phosphatidic acid (in the absence of ethanol). Phorbol esters as a second stimulus with anti-CD3 antibody led to a additive increase in cellular diacylglycerol content but provided no increased production of inositol phosphates, suggesting that diacylglycerol production in these cells results from hydrolysis of noninositol containing lipids as well as from phosphinositides. Exogenous addition of phosphatidic acid led to increases in cytosolic calcium that, depending on the concentration used, resulted from release of an intracellular store of calcium and influx of extracellular calcium. Changes in cytosolic calcium occurred in the absence of inositol phosphates production. These studies establish a role for increased phospholipase D activity in T lymphocyte activation.