Identification and cloning of the SNARE proteins VAMP-2 and syntaxin-4 from HL-60 cells and human neutrophils.

Degranulation and membrane fusion by neutrophils are essential to host defense. We sought homologues of neuron-specific fusion proteins in human neutrophils and in their precursors, the promyelocytic cell line HL-60. We screened a differentiated HL-60 library and obtained an 848 bp sequence with a 351 bp open reading frame, identical to that published for human VAMP-2 and including 5' and 3' untranslated regions. RNA from HL-60 cells during differentiation into the neutrophil lineage was subjected to Northern blot analysis. which revealed a transcript of approximately 1050 bp at all stages of differentiation. The amount of these transcripts increased approximately threefold during differentiation, a finding confirmed by quantitative RT-PCR. We also detected mRNA for VAMP-2 in human neutrophils and monocytes using RT-PCR. In like fashion, transcripts of syntaxin-4, another fusion protein, were recovered from a neutrophil cDNA library. As with VAMP-2, expression of syntaxin-4 (determined by Northern blots) also increased, but by only 50%, during differentiation of HL-60 cells. These studies demonstrate that neutrophils and their progenitors possess mRNA for the fusion proteins VAMP-2 and syntaxin-4, and that their transcription increases during differentiation, concurrent with the functional maturation of myeloid cells.

L-selectin signaling of neutrophil adhesion and degranulation involves p38 mitogen-activated protein kinase.

The adhesion molecules known as selectins mediate the capture of neutrophils from the bloodstream. We have previously reported that ligation and cross-linking of L-selectin on the neutrophil surface enhances the adhesive function of beta(2)-integrins in a synergistic manner with chemotactic agonists. In this work, we examined degranulation and adhesion of neutrophils in response to cross-linking of L-selectin and addition of interleukin-8. Cross-linking of L-selectin induced priming of degranulation that was similar to that observed with the alkaloid cytochalasin B. Activation mediated by L-selectin of neutrophil shape change and adhesion through CD11b/CD18 were strongly blocked by Merck C, an imidazole-based inhibitor of p38 mitogen-activated protein kinase (MAPK), but not by a structurally similar non-binding regioisomer. Priming by L-selectin of the release of secondary, tertiary, and secretory, but not primary, granules was blocked by inhibition of p38 MAPK. Peak phosphorylation of p38 MAPK was observed within 1 min of cross-linking L-selectin, whereas phosphorylation of ERK1/2 was highest at 10 min. Phosphorylation of p38 MAPK, but not ERK1/2, was inhibited by Merck C. These data suggest that signal transduction as a result of clustering L-selectin utilizes p38 MAPK to effect neutrophil shape change, integrin activation, and the release of secondary, tertiary, and secretory granules.

Antiorthostatic suspension for 14 days does not diminish the oxidative response of neutrophils in mice.

The effects of long-term spaceflight on inflammatory responses have not been well-studied in either humans or animals. It is thus important to determine if the functions of immune and inflammatory cells are altered in models of spaceflight. One such animal model is antiorthostatic suspension (AOS), in which the experimental animal is subjected to a head-down tilt that mimics both the stress and the cephalad fluid shift experienced in spaceflight. A previous study reported that the peritoneal neutrophils from mice experiencing AOS generated less superoxide than unsuspended controls. We expanded on this study using several different stimuli and measuring the oxidative response of murine neutrophils in a variety of ways. These responses included the rate, lag period, and dose/response characteristics for superoxide generation, FACS analysis with dihydrodichlorofluorescein as a substrate, and a chemiluminescence response with luminol as a substrate. We also examined phagocytosis of three different microorganisms. While some effects of orthostatic suspension (attributable to the stress of the apparatus) were observed, no clear effects of AOS on oxidative function of the peritoneal neutrophils were seen.

Identification of glyceraldehyde-3-phosphate dehydrogenase as a Ca2+-dependent fusogen in human neutrophil cytosol.

The membrane fusion events observed during neutrophil degranulation are important aspects of the immunoregulatory system. In an attempt to understand the regulation of granule-plasma membrane fusion, we have begun characterizing human neutrophil cytosol for fusion activity, finding that 50% of the fusogenic activity could be attributed to members of the annexin family of proteins. The major non-annexin fusion activity (25% of the total cytosolic activity) was enriched by ion exchange chromatography after depletion of annexins by Ca2+-dependent phospholipid affinity chromatography. The fusion activity co-purified with a 10,14-kDa dimer identified as leukocyte L1 (which was non-fusogenic), along with an approximately 36-kDa protein. This protein was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by amino-terminal sequencing, and the fusion activity was verified using commercially available GAPDH. GAPDH may play an important role in degranulation because it is as potent as annexin I on a mass basis and may constitute up to 25% of the total cytosolic fusion activity of the neutrophil.

Phospholipase D activity facilitates Ca2+-induced aggregation and fusion of complex liposomes.

Phospholipase D (PLD) activation in stimulated neutrophils results in the conversion of membrane phosphatidylcholine (PC) to phosphatidic acid (PA). This change in membrane phospholipid composition has two potentially positive effects on degranulation. It 1) replaces a nonfusogenic phospholipid with a fusogenic one and 2) increases the potential for interactions between membranes and the annexins. Modeling neutrophil degranulation, we examined the effect of PLD (Streptomyces chromofuscus) hydrolysis on the aggregation and fusion of liposomes in the presence and absence of annexin I. We found that PLD-mediated conversion of PC to PA lowered the [Ca2+] required for fusion. Annexin I increased the rate of fusion in the presence of PA, although it did not lower threshold [Ca2+], which remained above the physiological range. However, after hydrolysis by PLD, annexin I lowered the [Ca2+] required for aggregation by almost three orders of magnitude, to near physiological concentrations. These studies indicate that the activation of PLD and the production of PA may play a role in annexin-mediated membrane-membrane apposition.

PLA2 promotes fusion between PMN-specific granules and complex liposomes.

Neutrophil stimulation results in the activation of a variety of phospholipases, including phospholipase A2 (PLA2), which releases arachidonic acid from the 2 position of membrane phospholipids, leaving a lysophospholipid. Because arachidonic acid is known to be a potent fusogen in vitro, we examined the effect of metabolism by PLA2 on the fusion of complex liposomes (liposomes prepared with a phospholipid composition similar to that found in neutrophil plasma membrane). We observed that PLA2 augmented the fusion of complex liposomes with each other as well as with specific granules isolated from human neutrophils, lowering the Ca2+ requirement for fusion by three orders of magnitude. Furthermore, although lysophospholipids inhibited fusion, the incorporation of arachidonic acid into liposome membranes overcame the inhibitory effects of the lysophospholipids. Thus with PLA2 and annexins we were able to obtain fusion of complex liposomes at concentrations of Ca2+ that are close to physiological. Our data suggest that the activation of PLA2 and the generation of arachidonic acid may be the major fusion-promoting event mediating neutrophil degranulation.

Development of an aqueous-space mixing assay for fusion of granules and plasma membranes from human neutrophils.

Several models have been developed to study neutrophil degranulation. At the most basic level, phospholipid vesicles have been used to investigate the lipid interactions occurring during membrane fusion. The two major forms of assays used to measure phospholipid vesicle fusion are based either on the dilution of tagged phospholipids within the membrane of the two fusing partners or the mixing of the aqueous contents of the vesicles. Although problems exist with both methods, the latter is considered to be more accurate and representative of true fusion. Using 8-aminonaphthalene-1,3,6-trisulphonic acid (ANTS) as a fluorescent marker, we have taken advantage of the quenching properties of p-xylenebispyridinium bromide ('DPX') to develop a simple aqueous-space mixing assay that can be used with any sealed vesicle. We compared our new assay with more conventional assays using liposomes composed of phosphatidic acid (PA) and phosphatidylethanolamine (PE), obtaining comparable results with respect to Ca2+-dependent fusion. We extended our studies to measure the fusion of neutrophil plasma-membrane vesicles as well as azurophil and specific granules with PA/PE (1:3) liposomes. Both specific granules and plasma-membrane vesicles fused with PA/PE liposomes at [Ca2+] as low as 500 microM, while azurophil granules showed no fusion at [Ca2+] as high as 12 mM. These differences in the ability of Ca2+ to induce fusion may be related to differences observed in whole cells with respect to secretion.

Modeling degranulation with liposomes: effect of lipid composition on membrane fusion.

Degranulation involves the regulated fusion of granule membrane with plasma membrane. To study the role of lipid composition in degranulation, large unilamellar vesicles (LUVs) of increasing complexity in lipid compositions were constructed and tested for Ca(2+)-mediated lipid and contents mixing. Lipid-mixing rates of LUVs composed of phosphatidylethanolamine (PE) and phosphatidylserine (PS) were strongly decreased by the addition of either phosphatidylcholine (PC) or sphingomyelin (SM), while phosphatidylinositol (PI) had little effect. "Complex" LUVs of PC:PE:SM:PI:PS (24:27:20:16:13, designed to emulate neutrophil plasma membranes) also showed very low rates of both lipid mixing and contents mixing. The addition of cholesterol significantly lowered the Ca2+ threshold for contents mixing and increased the maximum rates of both lipid and contents mixing in a dose-dependent manner. Membrane remodeling, which occurs in neutrophil plasma membranes upon stimulation, was simulated by incorporating low levels of phosphatidic acid (PA) or a diacylglycerol (DAG) into complex LUVs containing 50% cholesterol. The addition of PA both lowered the Ca2+ threshold and increased the rate of contents mixing in a dose-dependent manner, while the DAG had no significant effect. The interaction of dissimilar LUVs was also examined. Contents-mixing rates of LUVs of two different cholesterol contents were intermediate between the rates observed for the LUVs of identical composition. Thus, cholesterol needed to be present in only one fusing partner to enhance fusion. However, for PA to stimulate fusion, it had to be present in both sets of LUVs. These results suggest that the rate of degranulation may be increased by a rise in the cholesterol level of either the inner face of the plasma membrane or the outer face of the granule membrane. Further, the production of PA can promote fusion, and hence degranulation, whereas the subsequent conversion of PA to DAG may reverse this promotional effect.

Human neutrophil annexin I promotes granule aggregation and modulates Ca(2+)-dependent membrane fusion.

The mechanism and cofactor requirements of exocytotic membrane fusion in neutrophils are unknown. Cytosolic proteins have been implicated in membrane fusion events. We assessed neutrophil cytosol for the presence of fusogenic proteins using a liposome fusion assay (lipid mixing). A fusogenic 36-kD protein containing amino acid sequence homology with human annexin I was purified from the cytosol of human neutrophils. This protein also shared functional characteristics with annexin I: it associated with and promoted lipid mixing of liposomes in a Ca(2+)-dependent manner at micromolar Ca2+ concentrations. The 36-kD protein required diacylglycerol to promote true fusion (contents mixing) at the same Ca2+ concentrations used for lipid mixing. The 36-kD protein exhibited a biphasic dose-response curve, by both promoting and inhibiting Ca(2+)-dependent lipid-mixing between liposomes and a plasma membrane fraction. The 36-kD protein also promoted Ca(2+)-dependent increases in aggregation of a specific granule fraction, as measured by a turbidity increase. Antiannexin I antibodies depleted the 36-kD protein from the cytosol by greater than 70% and diminished its ability to promote lipid mixing. Antiannexin I antibodies also decreased by greater than 75% the ability of neutrophil cytosol to promote Ca(2+)-dependent aggregation of the specific granules. These data suggest that annexin I may be involved in aggregation and fusion events in neutrophils.

'Depletion' of ATP by 2-deoxyglucose: secretion by electroporated human neutrophils is not restored by readdition of ATP.

Studies of intracellular signal transduction are facilitated by the use of permeabilized cell systems, which permit the ready manipulation of the cytosol. These model systems have helped to define the roles that small solutes, particularly Ca2+ and nucleotides, play in stimulus-response coupling. In circumstances where the full depletion of intracellular ATP contents is required, some investigators have resorted to prior treatment with metabolic toxins, with the expectation that the role of ATP in signal transduction could then be more unambiguously studied. However, in the work reported here, we found that treatment with 2-deoxyglucose (2-DOG) irreversibly altered the cells: when poisoned human neutrophils were then permeabilized, the cells failed to degranulate well in response to Ca2+, and their sensitivity to Ca2+ could not be recovered by the readdition of ATP. Inhibition of secretion by 2-DOG was most pronounced when low concentrations of Ca2+ were used as the stimulus. Preincubation of the cells with only 1 mM 2-DOG for 10 min at 37 degrees C (prior to washing and permeabilizing the cells) was sufficient for maximal inhibition. Even without preincubation, high concentrations of 2-DOG directly inhibited secretion. The refractory nature of poisoned cells was not restored by the presence of Mg2+ and/or ATP. The protein kinase C agonist phorbol myristate acetate also did not restore sensitivity of secretion to Ca2+. Addition of ATP and/or GTP to the permeabilization medium (to maximize penetration of the nucleotides) failed to restore sensitivity; tracer studies demonstrated that these conditions were adequate for repletion of the nucleotide pool. These data indicate that human neutrophils poisoned with 2-DOG were irreversibly altered, such that restoration of the putative deficiency (ATP) was without effect. Experiments in which such preincubation measures are employed should be viewed with caution.

Dual effects of guanosine 5'-[gamma-thio]triphosphate on secretion by electroporated human neutrophils.

It is generally believed that G-proteins play stimulatory roles on cell activation. In contrast, we found that guanosine 5'-[gamma-thio]triphosphate (GTP[S]) was a potent inhibitor of Ca(2+)-induced secretion from specific granules (as monitored by vitamin B-12-binding protein). GTP[S] inhibition of specific-granule release occurred in the presence or absence of adenine nucleotides, required Mg2+ (1-3 mM), and was half-maximal at 30 microM-GTP[S]. The dual stimulatory and inhibitory effects of GTP[S] could be readily observed and differentiated when degranulation was monitored over a range of Ca2+ concentrations. Inhibition of specific-granule release by GTP[S] was observed at low Ca2+ concentrations and resulted from shifting the Ca2+ dose-response curves to the right. In contrast, GTP[S] promoted azurophil-granule secretion at relatively high concentrations of Ca2+ and appeared to be due to a general enhancement at all Ca2+ concentrations. A series of hydrolysable and non-hydrolysable nucleotides did not mimic GTP[S] or block its action. Inhibition by GTP[S] occurred in cells which were sensitized with a protein kinase C agonist, suggesting that inhibition of secretion took place distal to this enzyme. However, the inhibitory effects of GTP[S] on specific-granule secretion were reversed by cytochalasin D, which prevents new microfilament formation; this compound also enhanced the stimulation of azurophil-granule release by GTP[S]. We also found that GTP[S] greatly increased the F-actin content of permeabilized neutrophils, whereas Ca2+ (to a lesser extent) decreased F-actin. These data are consistent with the hypothesis that at least two G-proteins are involved in regulating secretion: one which has been previously described as stimulating Ca(2+)-induced secretion (particularly from azurophil granules) and a second, possibly involved in promoting microfilament assembly, which inhibits the discharge of specific granules.

Cyclic AMP inhibits secretion from electroporated human neutrophils.

It has long been known that intracellular cAMP inhibits and cGMP enhances intact neutrophil function. However, these effects are modest and require relatively high concentrations of the cyclic nucleotides. We decided to re-examine the effects of cyclic nucleotides on Ca2(+)-induced secretion by electroporated cells. This system allowed us to bypass normal cell surface receptor-ligand interactions as well as to directly expose the intracellular space to native cyclic nucleotides. We found that concentrations of cAMP as low as 3 microM inhibited Ca2(+)-induced secretion; 30-300 microM cAMP was maximally inhibitory. cAMP was actually slightly more potent than dibutyryl cAMP, a membrane-permeant derivative. In contrast, cGMP was only slightly stimulatory at 3 microM and modestly inhibitory at 300 microM; dibutyryl cGMP was ineffective. A more detailed investigation of the effects of cAMP showed that inhibition was only obtained in the presence of Mg2+. Half-maximal inhibition by cAMP occurred at 10-30 microM. Inhibition by cAMP was achieved by shifting the Ca2+ dose-response curve for secretion to the right; this was observed for the release of both specific granules (vitamin B12 binding protein) and azurophil granules (B-glucuronidase). We previously showed that ATP could enhance Ca2(+)-induced secretion in the presence of Mg2+, apparently by interacting with a cell surface purine receptor. However, increasing concentrations of ATP could not overcome inhibition by cAMP; this suggested that cAMP acted at some site other than the purine receptor. Inhibition by cAMP was also less apparent in the presence of the protein kinase C agonist phorbol myristate acetate (PMA), suggesting that the cyclic nucleotide did not produce systemic desensitization of the neutrophils. In summary, these results demonstrate that low, physiologically relevant concentrations of cAMP can modulate neutrophil responsiveness.

Calcium-dependent fusion of the plasma membrane fraction from human neutrophils with liposomes.

A cell-free assay monitoring lipid mixing was used to investigate the role of Ca2+ in neutrophil membrane-liposome fusion. Micromolar concentrations of Ca2+ were found to directly stimulate fusion of inside-out neutrophil plasma membrane enriched fractions (from neutrophils subjected to nitrogen cavitation) with liposomes (phosphatidylethanolamine:phosphatidic acid, 4:1 molar ratio). In contrast, right-side-out plasma membranes and granule membranes did not fuse with liposomes in the presence of Ca2+. Similar results were obtained with two different lipid mixing assays. Fusion of the neutrophil plasma membrane-enriched fraction with liposomes was dependent upon the concentration of Ca2+, with threshold and 50% maximal rate of fusion occurring at 2 microM and 50 microM, respectively. Furthermore, the fusion was highly specific for Ca2+; other divalent cations such as Ba2+, Mg2+ and Sr2+ promoted fusion only at millimolar concentrations. Red blood cell (RBC) membranes were used in control studies. Ca2(+)-dependent fusion did not occur between right-side-out or inside-out RBC-vesicles and liposomes. However, if the RBC-vesicles were exposed to conditions which depleted spectrin (i.e., low salt), then Ca2(+)-dependent fusion was detected. Other quantitative differences between neutrophil and RBC membranes were found; fusion of liposomes with RBC membranes was most readily achieved with La3+ while neutrophil membrane-liposome fusion was most readily obtained with Ca2+. Furthermore, GTP gamma S was found to enhance Ca2(+)-dependent fusion between liposomes and neutrophil plasma membranes, but not RBC membranes. These studies show that plasma membranes (enriched fractions) from neutrophils are readily capable of fusing with artificial lipid membranes in the presence of micromolar concentrations of Ca2+.

Lipocortins are major substrates for protein kinase C in extracts of human neutrophils.

We have identified two major proteins in human neutrophils that are phosphorylated in vitro by protein kinase C (PKC) as lipocortins III and a fragment of a lipocortin-like 68-kDa protein. In electroporated cells, the 68-kDa protein was phosphorylated during stimulation of the cells with either FMLP or PMA. Lipocortins are of interest because of their Ca2(+)- and phospholipid-dependent actin binding properties and ability to inhibit phospholipase A2. Two crude fractions of enzymes and proteins exposed to [gamma-32]PATP in the presence of Ca2+, Mg2+, phosphatidylserine and 1,2-oleoyl-acetyl-rac-glycerol were analyzed by gel electrophoresis and autoradiography. A number of proteins in a detergent-free fraction, including proteins at 36 and 32 kDa, were phosphorylated in the presence of these cofactors. In contrast, only two major proteins (35 and 32 kDa) were phosphorylated in a detergent-extracted fraction. Phosphorylation of the 36, 35, and 32 kDa proteins required the presence of Ca2+, Mg2+, and phosphatidylserine in our soluble fraction and detergent extract, indicating PKC-dependent phosphorylation. The 32-kDa protein phosphorylated in both the soluble fraction and detergent extract was identified as lipocortin III by immunoprecipitation with a cross-reactive antibody that recognized lipocortin I and comparison of cyanogen bromide (CNBr) cleavage patterns of this protein with a lipocortin III standard. The 68-kDa protein was identified as a lipocortin VI-like protein by immunoprecipitation with anti-calelectrin. Additionally, the CNBr cleavage pattern of the 68-kDa protein was similar to that of the 36-kDa protein phosphorylated in our soluble fraction. Autoradiograms of the 68- and 36-kDa fragments immunoprecipitated from our soluble fraction with anticalelectrin and cleaved with CNBr showed that both of these proteins were phosphorylated in this sample. Because phosphorylation is known to change the functional characteristics of the lipocortins, the potential exists to link PKC and lipocortins in neutrophils to regulation of granulemembrane interactions or mediation of inflammation.

Ca2(+)-induced secretion by electropermeabilized human neutrophils. The roles of Ca2+, nucleotides and protein kinase C.

Studies of stimulus-response coupling have benefitted from the availability of permeabilization techniques, whereby putative second messengers and intracellular modulators can be introduced into the cell interior. Electropermeabilization, which uses high-intensity electric fields to breach the plasma membrane, creates small pores, permitting access of solutes with molecular masses below 700 KDa. Neutrophils permeabilized by this technique, but not intact cells, discharged lysosomal constituents when exposed to micromolar levels of Ca2+. Secretion by electroporated neutrophils was significantly enhanced by the presence of Mg-ATP (0.3-1.0 mM). Contrary to expectations, it was determined that ATP was not the only nucleotide which enhanced Ca2(+)-induced secretion in the presence of Mg2+. Not only could GTP, XTP, ITP, UTP or ADP partially or completely replace ATP, but even non-hydrolyzable nucleotides such as ADP beta S ATP gamma S, and App[NH]p were effective. GTP gamma S and GDP beta S were inhibitory, while Gpp[NH]p was inactive. None of these nucleotides induced secretion on its own. In contrast, neutrophils which were permeabilized and then washed, were only slightly activated by Mg-ATP and other nucleotides; even the response to Ca2+ alone was less. This hyporesponsiveness of washed cells proved to be due to a time-dependent deactivation of the permeabilized neutrophils taking place at 4 degrees C. In an effort to assess the role for protein kinase C (PKC) in secretion in this system, we examined the effects of phorbol myristate acetate (PMA), a PKC agonist. PMA enhanced degranulation induced by Ca2+ by lowering the requirement for this divalent cation; enhancement by PMA was not dependent upon exogenous ATP. Three inhibitors of PKC with varying specificity, namely H-7, K-252a, and staurosporine, all abrogated PMA-enhanced secretion. These agents also inhibited secretion stimulated by Ca2+ plus ATP in parallel with that induced by Ca2+ plus PMA, strongly suggesting a role for PKC in modulation of degranulation by ATP. Our results show that electropermeabilized neutrophils provide a convenient, useful model for stimulus-secretion coupling. These data also suggest that the 'requirement' for Mg-ATP, which has been observed in other permeabilized cell systems, is not simply for metabolic energy or as a substrate for kinases. It is possible that these nucleotides all interact with a recently described neutrophil receptor for adenine nucleotides or with a recently postulated exocytosis-linked G-protein.

Exposure of human neutrophils to exogenous nucleotides causes elevation in intracellular calcium, transmembrane calcium fluxes, and an alteration of a cytosolic factor resulting in enhanced superoxide production in response to FMLP and arachidonic acid.

Exposure of human neutrophils to micromolar concentrations of both hydrolyzable and nonhydrolyzable purine nucleotides caused the generation of transient rises in intracellular calcium (Ca2+), Ca2+ fluxes across the membrane, and primed the cells for enhanced production of superoxide (O2-) when subsequently exposed to agonists such as FMLP and arachidonic acid. The neutrophils were most sensitive to adenosine triphosphate (ATP) and ATP-gamma-S, which produced Ca2+ transients and enhanced O2- production at concentrations as low as 1 to 5 mumol/L, with a doubling of O2- generation at 25 to 50 mumol/L. Adenosine diphosphate (ADP), guanosine triphosphate (GTP), and 5'-adenylylimidodiphosphate (AMP-PNP) required approximately 10-fold higher concentrations to cause similar effects. Adenosine did not cause Ca2+ fluxes or a Ca2+ transient and was inhibitory of O2- production. There was a strong correlation between a nucleotide's ability to generate a Ca2+ response and its ability to enhance O2- generation. Nitrogen cavitation and subcellular fractionation of the neutrophils after a brief exposure to ATP, ATP-gamma-S, and AMP-PNP revealed that the enhanced O2- generating capacity was stable and detectable in a cell-free assay system. By combining variously treated cytosolic and membrane fractions, it was found that the enhanced O2- production was attributable to a modification of a component(s) of the cytosol.

Human neutrophils contain a protein kinase C-like enzyme that utilizes guanosine triphosphate as a phosphate donor. Cofactor requirements, kinetics, and endogenous acceptor proteins.

Investigations of protein kinase C (PKC) activity have focussed on protein phosphorylation using adenosine triphosphate (ATP), not guanosine triphosphate (GTP), as the phosphate donor. In a continuing study of the enzymology of the PKC of human neutrophils, we wanted to determine if there might be protein kinases that do use GTP as a phosphate donor. Soluble extracts or detergent-extracted fractions of human neutrophils were used as enzyme sources. Phosphorylation of histone using [gamma-32P]-GTP was 31% as effective as [gamma-32P]-ATP. Phosphorylation with GTP depended on Ca2+, Mg2+, and phospholipid, just as the ATP, and the Ca2+ requirements were similar. In all cases, H-7, an inhibitor of ATP-supported PKC activity, blocked GTP-utilizing activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that similar endogenous proteins were phosphorylated with ATP or GTP. The apparent Km and Vmax for the enzyme(s) for both phosphate donors were identical, although these were modified by treatment with Triton X-100. GTP competitively inhibited use of ATP by PKC; however, low concentrations of ATP enhanced GTP-utilizing kinase activity in some cases. Non-hydrolyzable forms of ATP and other nucleotide triphosphates were inhibitory. Detergent treatment also markedly altered the number of proteins phosphorylated by either nucleotide. The major protein phosphorylated in the soluble or detergent extract was a single polypeptide band in the 34 Kd range. These studies are the first to explicitly examine the possible phosphorylation by neutrophil PKC using GTP and point to a potential alternative mode of enzyme activity. Since high concentrations of GTP are available within neutrophils, the ability of PKC or a PKC-like enzyme to use this nucleotide may have important ramifications in signal transduction.

The effects of heavy metal cations and sulfhydryl reagents on degranulation from digitonin-permeabilized neutrophils.

Digitonin-permeabilized neutrophils were exposed to micromolar levels of a variety of heavy metal cations and sulfhydryl oxidants to gain insight into the potential biochemical mechanisms underlying neutrophil degranulation. The results from this study suggest that the oxidation of intracellular sulfhydryl groups may play a role in neutrophil signal transduction. Evidence to support this conclusion is based on the observation that cupric phenanthroline and Cu2+/cysteine, agents reported to induce disulfide bond formation, evoke significant granule enzyme release when presented to permeabilized neutrophils. The stimulatory actions of these compounds occur in the absence of Ca2+ and are blocked by the sulfhydryl reducing agent, dithiothreitol. In addition, we observed marked potentiation of Ca2+-induced secretion by potentially physiological levels of Ni2+. Although we are unaware of any Ni2+-requiring enzymes in eukaryotic cells that are likely to be pertinent to degranulation, the ability of this divalent metal cation to lower the Ca2+ requirements for granule secretion suggests that it may play an important regulatory role in Ca2+-dependent processes. Finally, we observed significant granule release when permeabilized neutrophils were exposed to the heavy metal cations, Hg2+ and Ag+. The apparent stimulatory actions of these metals were the result of lysis rather than degranulation. Thus, the ability of these metals to lyse intracellular organelles such as lysosomal granules may contribute to their toxicological properties.