Fc gamma receptor signal transduction in natural killer cells. Coupling to phospholipase C via a G protein-independent, but tyrosine kinase-dependent pathway.

Antibody-dependent cellular cytotoxicity is initiated when low affinity Fc receptors (Fc gamma R type III/CD16) on NK cells bind to sensitized (i.e., antibody coated) target cells. Fc gamma R cross-linkage induces the activation of phospholipase C (PLC), which hydrolyses membrane phosphoinositides, generating inositol-1,4,5-trisphosphate and sn-1,2-diacylglycerol as second messengers. However, the mechanism that couples Fc gamma R stimulation to PLC activation remains unknown. In this study, we investigated whether the Fc gamma R is coupled to PLC via a guanine nucleotide-binding (G) protein or an alternative pathway. Stimulation of electropermeabilized human NK cells with GTP gamma S induced inositol phosphate (IP) release, indicating the presence of a G protein-linked PLC activity in these cells. However, stimulation with both anti-Fc gamma R mAb and GTP gamma S provoked additive rather than synergistic increases in IP formation. Furthermore, exogenous GDP strongly inhibited GTP gamma S-stimulated IP release, but failed to inhibit the response to anti-Fc gamma R mAb stimulation. These results suggested GTP gamma S and anti-Fc gamma R mAb activated PLC through distinct regulatory mechanisms, and that Fc gamma R was not linked to PLC via a G protein. Hence, an alternative transduction mechanism for Fc gamma R-PLC coupling was considered. Antibody-mediated Fc gamma R cross-linkage was shown to rapidly stimulate tyrosine phosphorylation of multiple proteins in NK cells. Pretreatment with the tyrosine kinase inhibitor, herbimycin A, inhibited these phosphorylation events and disrupted the coupling between Fc gamma R ligation and PLC activation. These observations suggest that Fc gamma R in NK cell is coupled to PLC via a G protein-independent, but tyrosine kinase-dependent pathway.     

Role for the Rac1 exchange factor Vav in the signaling pathways leading to NK cell cytotoxicity

Here we investigate the activation of and a possible role for the hematopoietic Rac1 exchange factor, Vav, in the signaling mechanisms leading to NK cell-mediated cytotoxicity. Our data show that direct contact of NK cells with a panel of sensitive tumor targets leads to a rapid and transient tyrosine phosphorylation of Vav and to its association with tyrosine-phosphorylated Syk. Vav tyrosine phosphorylation is also observed following the activation of NK cells through the low-affinity Fc receptor for IgG (Fc gamma RIII). In addition, we demonstrate that both direct and Ab-mediated NK cell binding to target cells result in the activation of nucleotide exchange on endogenous Rac1. Furthermore, Vav antisense oligodeoxynucleotide treatment leads to an impairment of NK cytotoxicity, with Fc gamma RIII-mediated killing being more sensitive to the abrogation of Vav expression. These results provide new insight into the signaling pathways leading to cytotoxic effector function and define a role for Vav in the activation of NK cell-mediated killing.     

The low affinity 40,000 Fc gamma receptor and the transferrin receptor can be alternative or simultaneous target structures on cells sensitive for natural killing

The role of the low avidity 40,000 dalton receptor for IgG (Fc gamma R) present on K562 and U937 cells in sensitivity to natural killing (NK) was studied by using a murine monoclonal antibody (mAb) specific for the 40,000 dalton Fc gamma R (alpha Fc gamma R mAb). Pretreatment of K562 target cells with intact alpha Fc gamma R mAb or its Fab fragment or anti-transferrin receptor (alpha TFR) mAb partially blocked in a dose-dependent manner, NK activity to K562 cells. However, combined pretreatment with alpha Fc gamma R and alpha TFR mAb completely blocked NK activity against K562 targets. As compared with K562 cells, lower levels of NK were elicited against Molt-4, U937, HL-60, and Daudi targets. Although NK activity to Molt-4 targets was not affected by alpha Fc gamma R mAb, it was fully prevented by pretreatment with alpha TFR mAb. In contrast, NK to U937 cells was not influenced by alpha TFR mAb, but it was strongly inhibited by alpha Fc gamma R mAb. The resistance of 3H-TdR-prelabeled adherent HEp-2 cells to natural cell-mediated cytotoxicity was not affected by either mAb. Lectin-dependent cell-mediated cytotoxicity (LDCC) against HEp-2 cells due to the presence of concanavalin A, and was completely abrogated by pretreatment of the targets with alpha TFR mAb, but was unaffected by alpha Fc gamma R mAb. By use of the flow cytometer, a significant correlation was detected between the relative expression of 40,000 dalton Fc gamma R and the susceptibility to NK, whereas the expression of TFR was discordant from NK sensitivity. As determined in the single cell cytotoxicity assay alpha Fc gamma R mAb reduced the frequency of target binding effector cells without affecting the number of dead bound targets. This pattern of inhibition was found against both K562 and U937 targets. Alternatively, alpha TFR mAb inhibited both binding and killing of K562 and Molt-4 targets. Because pretreatment of HEp-2 cells with alpha TFR mAb did not influence conjugate formation, the blocking of LDCC to HEp-2 cells by alpha TFR mAb can be related to post-binding events. These data show that although both the 40,000 dalton Fc gamma R and the TFR can be target structures for NK cell recognition, the TFR may also play an important role in the post-binding events.     

Activation of cloned human natural killer cells via Fc gamma RIII

The Fc gamma RIII (CD16) Ag on human NK cells involved in antibody-dependent cellular cytotoxicity has been demonstrated to be an important activation structure. The present studies were carried out to further characterize the functional role of the CD16 Ag and the mechanisms whereby cytotoxicity is activated by using human NK clones. In phenotypic studies Fc gamma RIII was found to be expressed heterogeneously on various human cloned NK cells. Expression on CD3- and CD3+ clones varied with the donor and mAb used for detection. Functional data demonstrated that cytotoxicity against NK-resistant target cells can be induced in CD3-CD16+ NK clones and CD3+CD16+ clones with NK activity when various CD16 mAb were used. CD16 antibodies but not reactive isotype control antibodies induced cytotoxicity. In contrast to complete CD16 antibodies F(ab')2 fragments were not able to activate the cytotoxic mechanism. Both an antibody against FcR on the target cell (Fc gamma RII) and a CD11a antibody blocked induction of cytotoxicity. These results suggest that three steps are critical for activation of CD16+ cells via Fc gamma RIII: 1) specific binding of CD16 antibodies to Fc gamma RIII on effector cells irrespective of the epitope recognized; 2) cross-linking of effector cell CD16 Ag through binding of the Fc site of CD16 antibodies via corresponding FcR on the target cell membrane; and 3) interaction of CD11a/18 molecules with the target cell membrane.     

Involvement of pertussis toxin-sensitive and -insensitive GTP-binding proteins in luteinizing hormone exocytosis distal to second messenger generation.

Inhibition of luteinizing hormone (LH) exocytosis by guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) in permeabilized pituitary cells has indicated the involvement of one or more GTP-binding proteins in the exocytotic mechanism distal to second messenger generation. We now report that two inhibitory sites of action of GTP gamma S can be distinguished by their dependence on GTP gamma S concentration and their sensitivity to pertussis toxin. Ca(2+)-stimulated exocytosis was half-maximally inhibited by 6.8 microM GTP gamma S, a six-fold higher concentration than that required for inhibition of exocytosis stimulated by phorbol ester plus cAMP. In addition, GTP gamma S inhibition of Ca(2+)-stimulated exocytosis was insensitive to pertussis toxin, in contrast to the inhibition of exocytosis stimulated by phorbol ester plus cAMP, which was abolished by pretreatment with pertussis toxin. These results indicate that at least two stimulus-specific GTP-binding proteins are involved in regulating LH exocytosis distal to second messenger generation.     

Differentiation-linked activation of the respiratory burst in a monocytic cell line (U937) via Fc gamma RII. A study of activation pathways and their regulation.

Activation of the respiratory burst in the monocytic cell line U937 by cross-linking human 40-kDa FcR for IgG (Fc gamma RII) with the IgG1 mAb, CIKM5, is dependent on the maturation state of the cell. Addition of anti-Fc gamma RII to undifferentiated cells does not activate the respiratory burst but differentiation with human rIFN-gamma (200 U/ml) for 13 to 15 days results in maximal stimulation by this agonist, with half-maximal responses in cells incubated for 10 to 12 days. During maturation the development of responsiveness to cross-linking Fc gamma RII occurs later than the development of responsiveness to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (maximal responses at 7 to 9 days), or the chemotactic peptide FMLP (half-maximal responses at 7 to 9 days). The late development of maximal Fc gamma RII responses is not associated with either increased Fc gamma RII expression, enhanced calcium mobilization induced by anti-Fc gamma RII, changes in protein kinase C activity (PKC) or a switch in PKC isotype expression. Activation of the respiratory burst via Fc gamma RII may not be mediated by activation of PKC as the kinase inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride and N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride inhibited the Fc gamma RII response by less than 20% at concentrations which inhibit the 12-O-tetradecanoylphorbol-13-acetate-induced respiratory burst by more than 80%. IFN-gamma U937 cells did not metabolize incorporated arachidonate into eicosanoids when stimulated with anti-Fc gamma RII, suggesting that eicosanoids do not mediate activation of the respiratory burst, and this was confirmed by the lack of inhibition by the specific 5'-lipoxygenase and glutathione S-transferase inhibitor, piriprost, and the cyclo-oxygenase inhibitor, indomethacin. In addition there was no significant release of radiolabeled arachidonate in response to anti-Fc gamma RII. The response to anti-Fc gamma RII is inhibited by pertussis toxin, suggesting that signal transduction is via a GTP-binding protein. Agents that elevate intracellular cAMP increased the magnitude of the cAMP transients stimulated by anti-Fc gamma RII and also inhibited the respiratory burst. FMLP responses showed a similar pattern of sensitivity to this range of inhibitors, suggesting that both Fc gamma RII and FMLP receptor share common regulatory mechanisms. However, the termination of the respiratory burst activated via Fc gamma RII and FMLP receptor is independently regulated, in that after FMLP-induced activation there is no subsequent inhibition of the Fc gamma RII-mediated response and vice versa.     

The epidermal growth factor receptor is coupled to a phospholipase A2-specific pertussis toxin-inhibitable guanine nucleotide-binding regulatory protein in cultured rat inner medullary collecting tubule cells

Studies were performed to examine a potential role for a guanine nucleotide-binding protein in epidermal growth factor (EGF)-stimulated phospholipase A2 (PLA2) activity. EGF increased prostaglandin E2 (PGE2) production in intact or saponin-permeabilized rat inner medullary collecting tubule (RIMCT) cells. Incubation of permeabilized cells with guanosine 5'-O-(thiotriphosphate) (GTP gamma S) enhanced and with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) inhibited the response to EGF. GDP beta S had no effect on ionomycin-stimulated PGE2 production. Exposure of intact cells to 25 mM NaF + 10 microM AlCl3 enhanced both basal and EGF-stimulated PGE2 production. Pertussis toxin ADP-ribosylated a 41-kDa protein in RIMCT cell membranes. Pretreatment of cells with pertussis toxin (100 ng/ml for 16 h) eliminated the response to EGF in intact cells and the response to EGF + GTP gamma S in permeabilized cells. Pertussis toxin had no effect on the response to ionomycin. The effect of pertussis toxin was not due to alterations in cAMP as cellular cAMP levels were unaffected by pertussis toxin both in the basal state and in the presence of EGF. PGE2 production in response to EGF was not transduced by a G protein coupled to phospholipase C (PLC) as neomycin, which inhibited PLC, did not decrease EGF-stimulated PGE2 production. Also, PGE2 production was not increased by inositol trisphosphate and did not require the presence of extracellular Ca2+. In contrast to EGF-stimulated PLC activity, stimulation of PLA2 by EGF was not susceptible to inhibition by phorbol 12-myristate 13-acetate. These results clearly demonstrate the existence of a PLA2-specific pertussis toxin-inhibitable guanine nucleotide-binding protein coupled to the EGF receptor in RIMCT cells.     

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.     

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.     

Cross-linking of the high affinity Fc receptor for human immunoglobulin G1 triggers transient activation of NADPH oxidase activity. Continuous oxidase activation requires continuous de novo receptor cross-linking

Cross-linking of the high affinity Fc receptor for human immunoglobulin G1 (Fc gamma RI) on U937 cells triggered superoxide anion (O-2) release. This was accomplished by the binding of an Fc gamma RI-specific monoclonal antibody, mAb 32, followed by cross-linking of the mAb on the cell with anti-mouse IgG F(ab')2 by Fc gamma RI-specific mAbs 32 and 22 used as an equimolar mixture or by Fc gamma RI-specific mAb 197 (a murine IgG2a and thus a multivalent ligand for Fc gamma RI) alone. At subsaturating concentrations of the Fc gamma RI-cross-linking ligands, O2- generation was continuous over relatively long intervals. However, saturating concentrations triggered an often substantial but always transient O2- burst. This transient burst of oxidase activity ceased with maximal ligand accumulation on the cell. Cells in which oxidase activity had ceased could be restimulated using phorbol 12-myristate 13-acetate or aggregated human IgG1, indicating that cessation of O2- generation was not due to a generalized exhaustion or inhibition of the NADPH oxidase pathway. Cells incubated in subsaturating concentrations of cross-linking antibodies continued to release O2- until binding of the ligand ceased. In addition, the rates of O2- production and ligand accumulation were the same. Thus, continuous O2- production appeared to be dependent upon continuous de novo formation of cross-linked and activated Fc gamma RI. Furthermore, the mol of O2- released in response to Fc gamma RI cross-linking by the multivalent ligand mAb 197 were directly proportional to the mol of mAb bound over a range of saturating and subsaturating concentrations. This evidence suggests a quantal relationship between each Fc gamma RI activated (cross-linked) and the resultant oxidase activity and supports a "rate" model for the activation of this response. Thus, each Fc gamma RI entering the pool of activated receptors probably makes a unitary contribution to the signal. An additional finding showed that cross-linked Fc gamma RI became associated with the cell cytoskeleton and that this association was also transient. Dissociation of Fc gamma RI from its cytoskeletal attachment occurred well after cessation of O2- production.     

Proliferative effect of PGD2 on osteoblast-like cells; independent activation of pertussis toxin-sensitive GTP-binding protein from PGE2 or PGF2 alpha.

PGD2 stimulated DNA synthesis and decreased alkaline phosphatase activity dose-dependently between 10 nM and 10 microM in osteoblast-like MC3T3-E1 cells. PGD2 had little effect on cAMP production, but caused very rapid enhancement of phosphoinositide (PI) hydrolysis dose-dependently between 10 nM and 10 microM. The formation of inositol trisphosphate (IP3) induced by PGD2 reached the peak within 1 min and decreased thereafter, which is more rapid than that induced by PGE2 or PGF2 alpha and both PGE2 and PGF2 alpha affected PGD2-induced IP3 formation additively. Pertussis toxin (PTX) inhibited both PGD2-induced formation of inositol phosphates and DNA synthesis. The degree of these PTX (1 micrograms/ml)-induced inhibitions was similar. In addition, neomycin, a phospholipase C inhibitor, inhibited PGD2-induced DNA synthesis as well as the formation of IP3, and the patterns of both inhibitions were similar. In the cell membranes, PTX-catalyzed ADP-ribosylation of a 40-kDa protein was significantly attenuated by pretreatment of PGD2. Time course of the attenuation of PTX-catalyzed ADP-ribosylation by PGD2 was apparently different from that by PGE2 or PGF2 alpha. These results indicate that PGD2 activates PTX-sensitive GTP-binding protein independently from PGE2 or PGF2 alpha and stimulates PI hydrolysis resulting in proliferation of osteoblast-like cells.     

Cross-talk regulation between cyclic AMP production and phosphoinositide hydrolysis induced by prostaglandin E2 in osteoblast-like cells.

In cloned osteoblast-like MC3T3-E1 cells, PGE2 stimulated both cAMP accumulation and the formation of inositol trisphosphate (IP3) dose dependently. The cAMP accumulation showed the peak value at 5 min and decreased thereafter, whereas the IP3 formation reached a plateau almost within 10 min and sustained it up to 30 min. The effect of PGE2 on cAMP accumulation (EC50 was 80 nM) was more potent than that on IP3 formation (EC50 was 0.8 microM). 12-O-Tetradecanoyl-phorbol-13-acetate (TPA), a protein kinase C (PKC)-activating phorbol ester, reduced the PGE2-induced cAMP accumulation, whereas 4 alpha-phorbol 12,13-didecanoate, a PKC-nonactivating phorbol ester, had little effect on the cAMP accumulation. 1-Oleoyl-2-acetyl-glycerol, a specific activator for PKC, inhibited PGE2-induced cAMP accumulation. TPA had little effect on cAMP accumulation induced by forskolin or NaF, a GTP-binding protein activator. So, the effect of TPA is presumed to be exerted at the point between the PGE2 receptor and Gs. On the other hand, forskolin and dibutyryl cAMP had little effect on the IP3 formation stimulated by PGE2. H-7, a PKC inhibitor, enhanced the PGE2-induced cAMP accumulation in comparison with HA1004, a control for H-7. Our data suggest that PGE2 regulates cAMP production through self-induced activation of PKC. These results strongly suggest that there is an autoregulatory mechanism in PGE2 signaling, and PGE2 modulates osteoblast functions through a cross-talk interaction between cAMP production and phosphoinositide hydrolysis in osteoblast-like cells.     

Coupling of the thyrotropin-releasing hormone receptor to phospholipase C by a GTP-binding protein distinct from the inhibitory or stimulatory GTP-binding protein

Thyrotropin-releasing hormone (TRH) stimulated a rapid rise in inositol trisphosphate (IP3) formation and prolactin release from 7315c tumor cells. The potencies (half-maximal) of TRH in stimulating IP3 formation and prolactin release were 100 +/- 30 and 140 +/- 30 mM, respectively. Pretreatment of the cells with pertussis toxin (for up to 24 h) had no effect on either process. Pretreatment of the cells with cholera toxin (30 nM for 24 h) also failed to affect basal or TRH-stimulated IP3 formation. TRH was also able to stimulate IP3 formation with a half-maximal potency of 118 +/- 10 nM in a lysed cell preparation of 7315c cells; the TRH-stimulated formation of IP3 was enhanced by GTP. 5'-Guanosine gamma-thiotriphosphate (GTP gamma S) and 5'-guanylyl imidodiphosphate (Gpp(NH)p), nonhydrolyzable analogs of GTP, stimulated IP3 formation in the absence of TRH with half-maximal potencies of 162 +/- 50 and 7500 +/- 4300 nM, respectively. In contrast to the lack of effect of pertussis toxin on the TRH receptor system, treatment of 7315c cells with pertussis toxin for 3 h or longer completely abolished the ability of morphine, an opiate agonist, to inhibit either adenylate cyclase activity or prolactin release. During this 3-h treatment, pertussis toxin was estimated to induce the endogenous ADP ribosylation of more than 70% of Ni, the inhibitory GTP-binding protein. GTP gamma S and Gpp(NH)p inhibited cholera toxin-stimulated adenylate cyclase activity (presumably by acting at Ni) with half-maximal potencies of 25 +/- 9 and 240 +/- 87 nM, respectively. Finally, Gpp(NH)p was also able to inhibit the [32P]ADP ribosylation of Ni with a half-maximal potency of 300 nM. These results suggest that a novel GTP-binding protein, distinct from Ni, couples the TRH receptor to the formation of IP3.     

Signal transduction during human natural killer cell activation: inositol phosphate generation and regulation by cyclic AMP

NK cells mediate both direct cytotoxicity against a variety of tumor cells and indirect (FcR-dependent) cytotoxicity against antibody-coated targets. When cloned human NK cells (CD16+/CD3-) were exposed to NK-sensitive targets for 30 min, the level of inositol phosphates rose two to five times above background. The rise in inositol phosphates induced by NK-sensitive targets was paralleled by an increase in intracellular free calcium concentration ([Ca2+]i). A panel of tumor cells that were resistant to NK cell lysis did not stimulate significant levels of inositol phosphate production and did not induce an elevation of intracellular free calcium. Ligation of the FcR (CD16) with the mAb 3G8 also triggered phosphoinositide turnover. Kinetic experiments demonstrated that stimulation by either susceptible target cells or by FcR ligation led to rapid (less than 1 min) generation of the Ca2+-mobilizing second messenger, inositol trisphosphate, with slower accumulation of inositol bisphosphate and inositol monophosphate. Previous studies have demonstrated that activation of the cAMP-dependent second messenger pathway strongly inhibits NK cell-mediated cytotoxic functions. Treatment of NK effector cells with forskolin to elevate intracellular cAMP levels resulted in a concentration-dependent inhibition of phosphoinositide hydrolysis induced by both NK-sensitive targets and 3G8-mediated FcR ligation. These results suggest that phosphoinositide turnover represents a critical early event in the human NK cell cytolytic process. Moreover, the potent inhibitory effect of cAMP on NK cell cytotoxicity may be explained by the uncoupling of NK receptors from phospholipase C-mediated phosphoinositide hydrolysis.     

Histamine-stimulated and GTP-binding proteins-mediated phospholipase A2 activation in rabbit platelets

Histamine is known to be a mediator of inflammation. In order to understand the role of histamine in platelets, we have examined the effects of histamine on arachidonic acid (AA) release, cAMP accumulation, inositol trisphosphate production, and serotonin secretion. Incubation of rabbit (and human) platelets with histamine resulted in rapid increase of [3H]AA release from the platelets prelabeled with [3H]AA. The effect of histamine was blocked by the addition of H1 receptor antagonist mepyramine. Histamine did not substantially affect the cAMP content and inositol trisphosphate production. Histamine-stimulated AA release was not observed in digitonin-permeabilized platelets, whereas histamine acted synergistically with GTP or GTP analog, guanosine 5'-(3-O-thio)triphosphate. Histamine-stimulated, and GTP analog-dependent AA release was inhibited by guanosine 5'-(2-O-thio) diphosphate. The effects of three receptor stimulants, thrombin, norepinephrine, and histamine were both diminished by 1 microgram/ml of pertussis toxin treatment and by the antiserum against GTP-binding proteins (G proteins) treatment. However, the antiserum against beta gamma subunits of G proteins inhibited the histamine effect, not thrombin effect. 4 beta-Phorbol 12-myristate 13-acetate (PMA) treatment enhanced histamine-stimulated AA release and serotonin secretion but inhibited thrombin-stimulated reactions. The effect of PMA was dose dependent and was due to enhance the coupling of histamine receptors and G proteins. The results show the existence of H1 histamine receptors which couple phospholipase A2 activation via pertussis toxin-sensitive G proteins. Histamine actions differ in sensitivities to anti-beta gamma antiserum treatment and PMA treatment from thrombin actions.     

Cholera toxin modulation of angiotensin II-stimulated inositol phosphate production in cultured vascular smooth muscle cells.

Activation of phospholipase C by angiotensin II in vascular smooth muscle has been postulated to be mediated by an unidentified GTP-binding protein (G-protein). Using a permeabilized preparation of myo-[3H]inositol-labelled cultured vascular smooth muscle cells, we examined the ability of a non-hydrolysable analogue of GTP, guanosine 5'-[gamma-thio]triphosphate (GTP[S]), to stimulate inositol phosphate formation. GTP[S] (5 min exposure) stimulated inositol polyphosphate release by up to 3.8-fold in a dose-dependent manner, with an EC50 (concn. producing half-maximal stimulation) of approx. 50 microM. Inositol bisphosphate (IP2) and inositol trisphosphate (IP3) accumulations were also stimulated by NaF (5-20 mM). Furthermore, angiotensin II-induced inositol phosphate formation could be potentiated by a submaximal concentration of GTP[S] (10 microM), and this treatment appeared to interfere with the normal termination mechanism of the initial hormonal signal. The G-protein mediating angiotensin II-stimulated phospholipase C activation was insensitive to pertussis toxin at an exposure time and concentration which were sufficient to completely ADP-ribosylate all available substrate (100 ng/ml, 16 h). In contrast, a similar incubation with cholera toxin markedly inhibited angiotensin II-stimulated IP2 and IP3 release by 67 +/- 6% and 62 +/- 6% respectively. Cholera toxin appeared to inhibit angiotensin II stimulation of phospholipase C by a dual mechanism: it caused a 45% decrease in angiotensin II receptor number, and also inhibited G-protein transduction as assessed by GTP[S]-stimulated IP2 formation. This latter inhibition may be secondary to an increase in cyclic AMP, since it could be simulated by addition of dibutyryl cyclic AMP. Thus angiotensin II-stimulated inositol phosphate formation is cholera-toxin-sensitive, and is mediated by a pertussis-toxin-insensitive G-protein, which may be involved directly in termination of early signal generation.     

Prostaglandin E receptors in bovine adrenal medulla are coupled to adenylate cyclase via Gi and to phosphoinositide metabolism in a pertussis toxin-insensitive manner

Prostaglandin E (PGE) receptor is coupled to a pertussis toxin-insensitive GTP-binding protein in bovine adrenal medulla, but PGE receptor partially purified from bovine adrenal medulla was functionally reconstituted with Gi into phospholipid vesicles (Negishi, M., Ito, S., Yokohama, H., Hayashi, H., Katada, T., Ui, M., and Hayaishi, O. (1988) J. Biol. Chem. 263, 6893-6900). We demonstrate here that PGE2 inhibited forskolin-induced accumulation of cAMP in cultured bovine chromaffin cells. In plasma membranes prepared from bovine adrenal medulla, PGE2 inhibited forskolin-stimulated adenylate cyclase activity in a GTP-dependent manner. This inhibitory action of PGE2 was abolished by treatment of the membrane with pertussis toxin. Reconstitution of the membranes ADP-ribosylated by pertussis toxin with Gi purified from bovine brain restored the potency of PGE2 to inhibit the adenylate cyclase activity. Inhibition of forskolin-induced cAMP accumulation by PGE2 was also abolished by exposure to the toxin in the cells, indicating that PGE receptors are coupled to Gi. In contrast, PGE2 stimulated the formation of inositol phosphates in chromaffin cells, but this effect was not affected by treatment of the cells with pertussis toxin, suggesting that the PGE receptors are coupled to phosphoinositide metabolism via a pertussis toxin-insensitive G-protein. Both the inhibitory action of cAMP accumulation and stimulation of phosphoinositide metabolism were specific for PGE1 and PGE2, and the Scatchard plot analysis of PGE2 binding to the membrane showed a single high-affinity binding site (Kd = 2 nM). In bovine adrenal chromaffin cells PGE2 enhanced catecholamine release in the presence of ouabain by stimulation of phosphoinositide metabolism (Yokohama, H., Tanaka, T., Ito, S., Negishi, M., Hayashi, H., and Hayaishi, O. (1988) J. Biol. Chem. 263, 1119-1122). We further examined the modulation of catecholamine release by PGE2 through its inhibitory coupling to the adenylate cyclase system. Prior exposure of chromaffin cells to forskolin or dibutyryl-cAMP reduced nicotine-stimulated catecholamine release, and PGE2 attenuated forskolin-induced inhibition of catecholamine release stimulated by nicotine, but not dibutyryl-cAMP-induced inhibition. In the absence of evidence that PGE receptor subtypes exist, these results suggest that the PGE receptor is coupled to two signal transduction systems leading to inhibition of cAMP accumulation via Gi and to production of inositol phosphates via a pertussis toxin-insensitive G-protein, both of which may modulate catecholamine release from bovine chromaffin cells.     

Evidence for coupling of bradykinin receptors to a guanine-nucleotide binding protein to stimulate arachidonate liberation in the osteoblast-like cell line, MC3T3-E1.

The effect of bradykinin on the activation production of inositol 1,4,5-trisphosphate and prostaglandin E2 (PGE2) was examined in the murine osteoblastic cell line, MC3T3-E1. Bradykinin, at concentrations ranging from 1 to 1000 nM, stimulated the production of inositol 1,4,5-trisphosphate 2.5- to 3-fold within 10 s, and elevated cytosolic-free Ca2+, even in the absence of external Ca2+. This process is mediated through the activation of phospholipase C. Bradykinin at the same concentration also stimulated the production of PGE2 and caused a release of 3H radioactivity from the cells prelabeled with [3H]arachidonic acid, probably via the activation of phospholipase A2. Pretreatment of the cells with pertussis toxin inhibited the stimulation of PGE2 production and 3H radioactivity release, while the elevation in cytosolic Ca2+ and the production of inositol 1,4,5-trisphosphate were not altered by toxin-pretreatment. The addition of an unhydrolyzable analog of GTP, guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) to the beta-escin-permeabilized cells prelabeled with [3H]arachidonic acid enhanced the release of 3H radioactivity. The simultaneous presence of bradykinin with GTP gamma S further activated the 3H radioactivity release in the beta-escin-permeabilized cells. These results provide evidence that receptors for bradykinin in the MC3T3-E1 couple stimulating arachidonate release, probably via the activation of phospholipase A2, through a guanine nucleotide binding protein sensitive to pertussis toxin.     

IgE-mediated activation of NK cells through Fc gamma RIII

NK cells express Fc gamma RIII (CD16), which is responsible for IgG-dependent cell cytotoxicity and for production of several cytokines and chemokines. Whereas Fc gamma RIII on NK cells is composed of both Fc gamma RIII alpha and FcR gamma chains, that on mast cells is distinct from NK cells and made of Fc gamma RIII alpha, FcR beta, and FcR gamma. Mast cells show degranulation and release several mediators, which cause anaphylactic responses upon cross-linking of Fc gamma RIII as well as Fc epsilon RI with aggregated IgE. In this paper, we examined whether IgE activates NK cells through Fc gamma RIII on their cell surface. We found that NK cells produce several cytokines and chemokines related to an allergic reaction upon IgE stimulation. Furthermore, NK cells exhibited cytotoxicity against IgE-coated target cells in an Fc gamma RIII-dependent manner. These effects of IgE through Fc gamma RIII were not observed in NK cells from FcR gamma-deficient mice lacking Fc gamma RIII expression. Collectively, these results demonstrate that NK cells can be activated with IgE through Fc gamma RIII and exhibit both cytokine/chemokine production and Ab-dependent cell cytotoxicity. These data imply that not only mast cells but also NK cells may contribute to IgE-mediated allergic responses.