Full-length and truncated neurokinin-1 receptor expression and function during monocyte/macrophage differentiation.

The substance P (SP)-preferring receptor neurokinin-1 receptor (NK-1R) has two forms: a full-length receptor consisting of 407 aa and a truncated receptor consisting of 311 aa. These two receptors differ in the length of the C terminus of NK-1R. We studied the undifferentiated and phorbol myristate acetate (PMA)-differentiated human monocyte/macrophage cell line THP-1 to investigate the expression and function of NK-1R. The expression of full-length and truncated NK-1R in this cell line was determined by using real-time PCR and immunofluorescence staining. Undifferentiated THP-1 cells expressed only truncated NK-1R. The differentiation of THP-1 cells with PMA to a macrophage-like phenotype resulted in the expression of full-length NK-1R, which was functionally accompanied by an SP (10(-6) M)-induced Ca2+ increase. In contrast, the addition of SP (10(-6) M) did not trigger Ca2+ response in undifferentiated THP-1 cells; however, SP did enhance the CCR5-preferring ligand RANTES (CCL5)-mediated Ca2+ increase. When a plasmid containing the full-length NK-1R was introduced into undifferentiated THP-1 cells, exposure to SP triggered Ca2+ increase, demonstrating that the full-length NK-1R is required for SP-induced Ca2+ increase. The NK-1R antagonist aprepitant (Emend, Merck) inhibited both the SP-induced Ca2+ increase in PMA-differentiated THP-1 cells and the SP priming effect on the CCL5-mediated Ca2+ increase, indicating that these effects are mediated through the full-length and truncated NK-1R, respectively. Taken together, these observations demonstrate that there are unique characteristics of NK-1R expression and NK-1R-mediated signaling between undifferentiated THP-1 cells and THP-1 cells differentiated to the macrophage phenotype.

Negative regulation of ligand-initiated Ca(2+) uptake by PKC-beta II in differentiated HL60 cells.

In phagocytic cells, fMet-Leu-Phe triggers phosphoinositide remodeling, activation of protein kinase C (PKC), release of intracellular Ca(2+) and uptake of extracellular Ca(2+). Uptake of extracellular Ca(2+) can be triggered by store-operated Ca(2+) channels (SOCC) and via a receptor-operated nonselective cation channel(s). In neutrophilic HL60 cells, the PKC activator phorbol myristate acetate (PMA) activates multiple PKC isotypes, PKC-alpha, PKC-beta, and PKC-delta, and inhibits ligand-initiated mobilization of intracellular Ca(2+) and uptake of extracellular Ca(2+). Therefore PKC is a negative regulator at several points in Ca(2+) mobilization. In contrast, selective depletion of PKC-beta in HL60 cells by an antisense strategy enhanced fMet-Leu-Phe-initiated Ca(2+) uptake but not mobilization of intracellular Ca(2+). Thapsigargin-induced Ca(2+) uptake through SOCC was not affected by PKC-beta II depletion. Thus PKC-beta II is a selective negative regulator of Ca(2+) uptake but not release of intracellular Ca(2+) stores. PKC-beta II inhibits a receptor-operated cation or Ca(2+) channel, thus inhibiting ligand-initiated Ca(2+) uptake.

TNFalpha elicits association of PI 3-kinase with the p60TNFR and activation of PI 3-kinase in adherent neutrophils.

Tumor necrosis factor (TNFalpha) is an incomplete secretagogue in neutrophils and requires the engagement of beta integrins to trigger secretion of superoxide anion (O(-)(2)). The p60 TNF receptor (p60TNFR) is responsible for signal transduction for activation of O(-)(2) generation. Activation of TNFalpha-triggered O(-)(2) generation in neutrophils adherent to fibrinogen-coated surfaces involves the beta2 integrin receptor CD11b/CD18. Phosphoinositide 3-kinase (PI 3-kinase) is essential for activation of O(-)(2) generation; wortmannin, an inhibitor of PI 3-kinase, inhibited TNFalpha-elicited O(-)(2) generation. p60TNFR immunoprecipitated from neutrophils was associated with immunoreactivity to PI 3-kinase in adherent neutrophils exposed to TNFalpha, but not in TNFalpha-treated neutrophils in suspension. In addition, PI 3-kinase immunoprecipitated from TNFalpha-activated neutrophils showed enhanced activity in adherent but not in nonadherent neutrophils. These findings suggest that synergism between CD11b/CD18 and p60TNFR in the presence of TNFalpha is required to elicit assembly of a signaling complex involving association of p60TNFR with PI 3-kinase, activation of PI 3-kinase, and generation of O(-)(2).

Glucagon-like peptide 1 stimulates lipolysis in clonal pancreatic beta-cells (HIT).

Glucagon-like peptide 1 (GLP-1) is the most potent physiological incretin for insulin secretion from the pancreatic beta-cell, but its mechanism of action has not been established. It interacts with specific cell-surface receptors, generates cAMP, and thereby activates protein kinase A (PKA). Many changes in pancreatic beta-cell function have been attributed to PKA activation, but the contribution of each one to the secretory response is unknown. We show here for the first time that GLP-1 rapidly released free fatty acids (FFAs) from cellular stores, thereby lowering intracellular pH (pHi) and stimulating FFA oxidation in clonal beta-cells (HIT). Similar changes were observed with forskolin, suggesting that stimulation of lipolysis was a function of PKA activation in beta-cells. Triacsin C, which inhibits the conversion of FFAs to long-chain acyl CoA (LC-CoA), enhanced basal FFA efflux as well as GLP-1-induced acidification and efflux of FFAs from the cell. Increasing the concentration of the lipase inhibitor orlistat progressively and largely diminished the increment in secretion caused by forskolin. However, glucose-stimulated secretion was less inhibited by orlistat and only at the highest concentration tested. Because the acute addition of FFAs also increases glucose-stimulated insulin secretion, these data suggest that the incretin function of GLP-1 may involve a major role for lipolysis in cAMP-mediated potentiation of secretion.

Roles for beta II-protein kinase C and RACK1 in positive and negative signaling for superoxide anion generation in differentiated HL60 cells.

beta-Protein kinase (PKC) is essential for ligand-initiated assembly of the NADPH oxidase for generation of superoxide anion (O(2)). Neutrophils and neutrophilic HL60 cells contain both betaI and betaII-PKC, isotypes that are derived by alternate splicing. betaI-PKC-positive and betaI-PKC null HL60 cells generated equivalent amounts of O(2) in response to fMet-Leu-Phe and phorbol myristate acetate. However, antisense depletion of betaII-PKC from betaI-PKC null cells inhibited ligand-initiated O(2) generation. fMet-Leu-Phe triggered association of a cytosolic NADPH oxidase component, p47(phox), with betaII-PKC but not with RACK1, a binding protein for betaII-PKC. Thus, RACK1 was not a component of the signaling complex for NADPH oxidase assembly. Inhibition of beta-PKC/RACK1 association by an inhibitory peptide or by antisense depletion of RACK1 enhanced O(2) generation. Therefore, betaII-PKC but not betaI-PKC is essential for activation of O(2) generation and plays a positive role in signaling for NADPH oxidase activation in association with p47(phox). In contrast, RACK1 is involved in negative signaling for O(2) generation. RACK1 binds to betaII-PKC but not with the p47(phox).betaII-PKC complex. RACK1 may divert betaII-PKC to other signaling pathways requiring beta-PKC for signal transduction. Alternatively, RACK1 may sequester betaII-PKC to down-regulate O(2) generation.

Serine phosphorylation of p60 tumor necrosis factor receptor by PKC-delta in TNF-alpha-activated neutrophils.

Tumor necrosis factor-alpha (TNF-alpha) triggers degranulation and oxygen radical release in adherent neutrophils. The p60TNF receptor (p60TNFR) is responsible for proinflammatory signaling, and protein kinase C (PKC) is a candidate for the regulation of p60TNFR. Both TNF-alpha and the PKC-activator phorbol 12-myristate 13-acetate triggered phosphorylation of p60TNFR. Receptor phosphorylation was on both serine and threonine but not on tyrosine residues. The PKC-delta isotype is a candidate enzyme for serine phosphorylation of p60TNFR. Staurosporine and the PKC-delta inhibitor rottlerin inhibited TNF-alpha-triggered serine but not threonine phosphorylation. Serine phosphorylation was associated with receptor desensitization, as inhibition of PKC resulted in enhanced degranulation (elastase release). After neutrophil activation, PKC-delta was the only PKC isotype that associated with p60TNFR within the correct time frame for receptor phosphorylation. In vitro, only PKC-delta, but not the alpha-, betaI-, betaII-, or zeta-isotypes, was competent to phosphorylate the receptor, indicating that p60TNFR is a direct substrate for PKC-delta. These findings suggest a selective role for PKC-delta in negative regulation of the p60TNFR and of TNF-alpha-induced signaling.

Long-chain acyl CoA regulation of protein kinase C and fatty acid potentiation of glucose-stimulated insulin secretion in clonal beta-cells.

Pancreatic beta-cells contain protein kinase C (PKC) isoforms that may play a role in insulin secretion. Activity of PKC classes (cPKC, nPKC, aPKC) and their regulation by acyl-CoA derivatives was examined in extracts of clonal pancreatic beta-cells (HIT) by protein phosphorylation. PKC classes were distinguished based on their previously defined cofactor requirements. Down-regulation of PKC by phorbol esters was confirmed by Western blotting and resulted in the complete loss of cPKC activity, partial loss of nPKC activity and preservation of aPKC activity and glucose-stimulated insulin secretion. aPKC activity was potentiated 4- to 8-fold by the CoA esters of myristate, palmitate, and oleate with a half-maximal value of 3 microM. Both oleoyl- and myristol-CoA, but not palmitoyl-CoA, caused inhibition of nPKC activity. Oleoyl-CoA inhibited nPKC activity up to 75% with a half-maximal effect at 10 microM. This value was independent of the concentration of diacylglycerol used. The addition of exogenous oleate or palmitate potentiated glucose-stimulated insulin secretion 2-fold and was unaffected by PMA-induced down-regulation. Stimulation by glucose or glucose and oleate also increased the mass of PKC-zeta found in the particulate fraction. These data are consistent with increased cytosolic long-chain acylCoA-activating aPKC isoforms resulting in stimulation and/or potentiation of glucose-induced insulin secretion.

Selective role for beta-protein kinase C in signaling for O-2 generation but not degranulation or adherence in differentiated HL60 cells.

A role for protein kinase C (PKC) isotypes is implicated in the activation of phagocytic cell functions. An antisense approach was used to selectively deplete beta-PKC, both betaI- and betaII-PKC, but not alpha-PKC, delta-PKC, or zeta-PKC in HL60 cells differentiated to a neutrophil-like phenotype (dHL60 cells). Depletion of beta-PKC in dHL60 cells elicited selective inhibition of O-2 generation triggered by fMet-Leu-Phe, immune complexes, or phorbol myristate acetate, an activator of PKC. In contrast, neither ligand-elicited beta-glucuronidase (azurophil granule) release nor adherence to fibronectin was inhibited by beta-PKC depletion. Ligand-induced phosphorylation of a subset of proteins was reduced in beta-PKC-depleted dHL60 cells. Phosphorylation of p47(phox) and translocation of p47(phox) to the membrane are essential for activation of the NADPH oxidase and generation of O-2. beta-PKC depletion had no effect on the level of p47(phox) in dHL60 cells but did significantly decrease ligand-induced phosphorylation of this protein. Furthermore, translocation of p47(phox) to the membrane in response to phorbol myristate acetate or fMet-Leu-Phe was reduced in beta-PKC-depleted cells. These results indicate that beta-PKC is essential for signaling for O-2 generation but not cell adherence or azurophil degranulation. Depletion of beta-PKC inhibited ligand-induced phosphorylation of p47(phox), translocation of p47(phox) to the membrane, and activation of O-2 generation.

Reversible Ca2+-dependent translocation of protein kinase C and glucose-induced insulin release.

It has been reported that protein kinase C (PKC) interacts at multiple sites in beta-cell stimulus-secretion coupling. Nevertheless, there is still controversy concerning the importance of this enzyme in glucose-induced insulin release. The present study was undertaken to clarify whether glucose, directly, or through changes in cytoplasmic free Ca2+ concentration, [Ca2+]i, could promote translocation of PKC from the soluble to the membrane compartment. Whereas glucose, which increases [Ca2+]i, did not affect long-term distribution of PKC activity between soluble and membrane fractions, this distribution was reversibly affected acutely by the Ca2+ concentration in the extraction media. Translocation of PKC to the membrane by incubation of HIT cells for 10 min in the presence of 20 nM phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in a 5-fold increase in glucose-induced insulin release. This was prevented by 50 nM concentration of the PKC inhibitor staurosporine, provided that the cells were exposed to the inhibitor before the phorbol ester. Cells pretreated with TPA demonstrated increased insulin secretion in response to glucose for several hours. This time course extended beyond the disappearance of [3H]TPA from the cells, which was complete after 1 h. Activation of PKC increased both average insulin release and the amplitude of oscillations 2-fold, but did not affect oscillation frequency. The stimulatory effect of increased PKC activity on insulin release was not matched by changes in [Ca2+]i. We suggest that stimulation of the pancreatic beta-cell with glucose promotes transient translocation of certain PKC isoforms from the cytoplasm to the plasma membrane as a direct consequence of the increase in [Ca2+]i. Such a translocation may promote phosphorylation of one or several proteins involved in the regulation of the beta-cell stimulus-secretion coupling. This results in potentiation of glucose-induced activation of insulin exocytosis, an effect then not mediated by an increase in [Ca2+]i per se. Hence, pulsatile insulin release can be obtained under conditions where overall [Ca2+]i does not change, challenging the view that oscillations in [Ca2+ ]i are indeed driving the oscillations in hormone release.

Cromolyn inhibits assembly of the NADPH oxidase and superoxide anion generation by human neutrophils.

Early and late phase reactions have been observed in asthma; the late phase reaction is characterized by accumulation of inflammatory cells such as neutrophils. Activated neutrophils degranulate and assemble an active NADPH oxidase, which generates superoxide anion (O2-), reactions that have been implicated in lung tissue damage. Preincubation of neutrophils with the asthma drug cromolyn sodium selectively inhibited FMLP (10(-7) M) and PMA (0.1 microgram/ml) elicited O2- generation but not degranulation. To further characterize the mechanism of this inhibition we examined the effect of cromolyn on the NADPH oxidase complex and the signaling pathways for its assembly. Ca2+ mobilization and activation of protein kinase C have been implicated as signals for activation of the NADPH oxidase. Ca2+ mobilization triggered by FMLP was significantly decreased by 21.2% in cromolyn-treated cells. In contrast, cromolyn did not interfere with translocation or activity of protein kinase C. Membranes prepared from neutrophils stimulated with 0.5 microgram/ml PMA generated O2-, indicating assembly of an active NADPH oxidase; cromolyn did not inhibit this membrane-associated, preassembled oxidase. In contrast, preincubation of neutrophils with 100 microM cromolyn before addition of PMA decreased the capacity of the membranes to generate O2- by 57.3%. These results indicate that cromolyn inhibited the assembly of an active NADPH oxidase. The efficacy of cromolyn may be associated with inhibition of assembly of an active NADPH oxidase in the neutrophil and prevention of oxygen radical-induced tissue damage.

Long chain acyl coenzyme A and signaling in neutrophils. An inhibitor of acyl coenzyme A synthetase, triacsin C, inhibits superoxide anion generation and degranulation by human neutrophils.

Ligand-initiated activation of neutrophils triggers O2- generation, degranulation, phospholipid remodeling, and release of fatty acids such as arachidonate, oleate, and palmitate. Long chain acyl-CoA synthetase converts free fatty acids to acyl-CoA esters; a role for acyl-CoA esters as positive modulators of neutrophil functions is proposed. Physiologically relevant concentrations (1-10 microM) of acyl-CoA esters such as palmitoyl-CoA, enhanced O2- generation triggered by fMet-Leu-Phe or guanosine 5'-O-(thiotriphosphate) (GTP gamma S) but did not act as a trigger per se. Triacsin C, an inhibitor of acyl-CoA synthetase, inhibited fMet-Leu-Phe-elicited O2- generation and degranulation in a concentration-dependent manner. Triacsin C inhibited O2- generation elicited by fMet-Leu-Phe and GTP gamma S in electroporated neutrophils, indicating that acyl-CoA acted downstream from the receptor. Palmitoyl-CoA reversed the Triacsin C-induced inhibition of O2- generation. fMet-Leu-Phe elicited a prompt increase in total long chain acyl-CoA esters. Arachidonoyl-CoA and oleoyl-CoA were elevated 5 s after addition of fMet-Leu-Phe, while palmitoyl-CoA was not elevated until 60 s. Triacsin C inhibited fMet-Leu-Phe-initiated increases in arachidonoyl-CoA, oleoyl-CoA, and palmitoyl-CoA. These results suggest a role for acyl-CoA esters in regulating activation of O2- generation and degranulation at the G protein or subsequent step(s).

Bovine parainfluenza-3 virus selectively depletes a calcium-independent, phospholipid-dependent protein kinase C and inhibits superoxide anion generation in bovine alveolar macrophages.

Bovine parainfluenza-3 (PI-3) virus inhibits oxygen-dependent bacterial killing by phagocytes, a key pulmonary defense, thus predisposing the host to intrapulmonary bacterial superinfection. PI-3 virus inhibited opsonized zymosan or PMA-activated superoxide anion (O2-) generation in bovine alveolar macrophages. The respiratory virus influenza also inhibits O2- generation by phagocytes, however, the mechanisms(s) of viral inhibition differs from PI-3. PI-3 did not trigger O2- generation before inhibition, whereas influenza triggered O2- generation before desensitization of ligand-initiated respiratory burst. PI-3 modified the twin signals of calcium and protein kinase C in alveolar macrophages. PI-3 infection increased macrophage membrane permeability to extracellular calcium, but did not inhibit calcium mobilization triggered by opsonized zymosan. These effects further distinguish bovine PI-3 from human influenza, which triggers mobilization of cell-associated calcium and inhibits calcium mobilization activated by physiologic ligands. Macrophages possessed two classes of PKC activity, a calcium/phosphatidylserine/diglyceride (Ca/PS/DG))-dependent activity and a Ca-independent, PS/DG-dependent histone IIIS phosphorylating activity. PI-3 infection selectively depleted the Ca-independent, PS/DG-dependent kinase activity but not the classical Ca/PS/DG-dependent activity. Inhibition of Ca-independent, PS/DG-dependent kinase activity and inhibition of O2- generation by PI-3 occurred at a similar viral dose and time frame, suggesting a role for this kinase in activating the respiratory burst. Inhibition of the oxygen-dependent bactericidal function of alveolar macrophages and disturbances in signal transduction may contribute to the immunosuppression and bacterial superinfection accompanying viral respiratory disease.

In vitro and in vivo effects of granulocyte colony-stimulating factor on neutrophils in glycogen storage disease type 1B: granulocyte colony-stimulating factor therapy corrects the neutropenia and the defects in respiratory burst activity and Ca2+ mobilization.

Children with glycogen storage disease (GSD) type 1b are susceptible to recurrent bacterial infections and have chronic neutropenia accompanied by phagocytic cell dysfunction including decreased superoxide anion (O2-) generation, calcium (Ca2+) mobilization, and chemotactic activity. Granulocyte colony-stimulating factor (G-CSF), a cytokine that corrects neutropenia in other diseases, in vitro enhances f-Met-Leu-Phe-triggered neutrophil O2- generation. Short-term pretreatment (15 min) of GSD 1b neutrophils with G-CSF increased the rate of O2- production (p < 0.01); however, this rate was still significantly below the rate of O2- production in control neutrophils. Recombinant human G-CSF (5 micrograms/kg/d) was administered s.c. to a GSD 1b patient. Before treatment, absolute neutrophil counts were < 500/mm3. Two d after G-CSF administration, the absolute neutrophil counts increased to 1333 and remained in the normal range during a 12-mo follow-up period. In vivo, G-CSF therapy increased f-Met-Leu-Phe-stimulated O2- production to 52% of control after 1 mo, and by mo 4, O2- production reached control levels. Our previous studies (J Clin Invest 56:196-202, 1990) demonstrated that decreased O2- production in neutrophils was associated with impaired Ca2+ mobilization. In vivo administration of G-CSF increased f-Met-Leu-Phe-triggered Ca2+ mobilization by neutrophils to 43% of control by mo 1 of G-CSF therapy and to 93% of control by mo 4, thus paralleling the improvements in O2- generation. In contrast, G-CSF therapy had no effect on the defective neutrophil chemotaxis. In summary, G-CSF therapy produced a rapid increase in circulating neutrophils and a gradual correction of O2- production.(ABSTRACT TRUNCATED AT 250 WORDS)

Interferon-gamma corrects the respiratory burst defect in vitro in monocyte-derived macrophages from glycogen storage disease type 1b patients.

Glycogen storage disease (GSD) type 1b is accompanied by decreased respiratory burst activity in peripheral blood phagocytic cells (i.e. monocytes and neutrophils). To elucidate whether this depressed respiratory burst was due to an intrinsic defect of phagocytic cells or due in part to in vivo host factors, we examined superoxide anion (O2-) production in monocytes from five GSD 1b patients cultured 9 d in vitro to allow for differentiation into macrophages (MDM). O2- production in MDM was measured in response to concanavalin A, fMet-Leu-Phe, and phorbol myristate acetate (PMA) stimulation. GSD 1b MDM had significantly depressed O2- generation with fMet-Leu-Phe and concanavalin A stimulation; however, unlike peripheral blood monocytes, GSD 1b MDM responded to PMA stimulation with O2- production comparable to healthy control donors. The cytokine interferon-gamma (IFN-gamma) has been shown to enhance O2- production in MDM. When GSD 1b MDM were cultured in the presence of IFN-gamma (1 x 10(5) U/L), O2- production in response to fMet-Leu-Phe, concanavalin A, and PMA was enhanced to rates similar to those of control MDM cultured in the presence of IFN-gamma. Thus, the respiratory burst defect observed in circulating phagocytic cells is also present in vitro in cultured GSD 1b MDM. However, in contrast to circulating phagocytic cells, depressed O2- production in GSD 1b MDM is selective to receptor-mediated activation, but not to PMA stimulation. This defect is correctable after short-term treatment with IFN-gamma, suggesting a role for IFN-gamma in treating the phagocytic defect in this disease.

Enhancement of macrophage candidacidal activity by interferon-gamma. Increased phagocytosis, killing, and calcium signal mediated by a decreased number of mannose receptors.

In contrast to its macrophage-activating capacity, IFN-gamma downregulates expression of the macrophage mannose receptor (MMR), which mediates uptake of Candida and other microorganisms. We found that IFN-gamma induced a concentration-dependent increase in the capacity of human monocyte-derived macrophages to ingest and kill both opsonized and unopsonized Candida albicans and to release superoxide anion upon stimulation with Candida. Mannan or mannosylated albumin inhibited this activated uptake of unopsonized Candida, but glucan did not. Addition of mAb to complement receptor (CR) 3 did not inhibit ingestion; macrophages that lacked CR3 (leukocyte adhesion defect) showed normal upregulation of ingestion by IFN-gamma. The increased candidacidal activity of IFN-gamma-activated macrophages was associated with reduced expression of MMR by a mean of 79% and decreased pinocytic uptake of 125I-mannosylated BSA by 73%; K(uptake) of pinocytosis was not changed. Exposure of resident macrophages to unopsonized Candida did not elicit a transient increase in intracellular free Ca2+ ([Ca2+]i); macrophages activated by IFN-gamma expressed a brisk increase in [Ca2+]i on exposure to Candida. These data suggest that macrophage activation by IFN-gamma can enhance resistance to C. albicans infection in spite of downregulation of the MMR, perhaps through enhanced coupling of the MMR to microbicidal functions.

Protein kinase C isotypes and signal-transduction in human neutrophils: selective substrate specificity of calcium-dependent beta-PKC and novel calcium-independent nPKC.

Neutrophils possess at least two phospholipid-dependent forms of protein kinase C, a classical Ca/PS/DG-dependent beta-isotype of protein kinase C and a Ca-independent but PS/DG-dependent novel protein kinase C (nPKC) which we now demonstrate to have different substrate specificities. Activation of human neutrophils triggers assembly of an NADPH oxidase in the membrane and generation of O2-. A role for the major Ca-dependent isotype beta-PKC in neutrophils is proposed in stimulus-induced phosphorylation and association of a cytosolic 47 kDa protein (p47-phox) with the membrane NADPH oxidase. In this study we demonstrate that purified beta-PKC and nPKC have very different substrate specificities; beta-PKC but not nPKC phosphorylated both endogenous and recombinant p47-phox. In addition, beta-PKC but not nPKC phosphorylated [ser25]PKC(19-31), the substrate peptide based on a sequence in the Ca-dependent alpha, beta and gamma-isotypes. Pseudosubstrate(19-36), derived from the C-terminus of Ca-dependent PKC isotypes, inhibited beta-PKC but not nPKC activity using either Histone IIIS or peptide(19-31) as substrate. Pseudosubstrate(19-36) also inhibited beta-PKC catalyzed phosphorylation of endogenous and recombinant p47-phox. Pseudosubstrate(19-36) also inhibited the O2- generation triggered by GTP gamma S in electroporated neutrophils by 50%. 32P-Labelled neutrophils electroporated in the presence of GTP gamma S showed phosphorylation of multiple cytosolic proteins including a 47 kDa band, and phosphorylation of membrane-associated 34 kDa, 47 kDa and 54 kDa proteins. Pseudosubstrate(19-36) inhibited phosphorylation of p47-phox in the membrane but not in the cytosol. These findings suggest translocatable, Ca-dependent isotypes of PKC such as beta-PKC may play a role in the phosphorylation of membrane associated p47-phox and the assembly or maintenance of an active NADPH oxidase.

Impairment of calcium mobilization in phagocytic cells in glycogen storage disease type 1b.

Patients with glycogen storage disease (GSD) type 1b, in contrast to patients with GSD 1a, are susceptible to recurrent bacterial infections suggesting defective phagocytic function. We have demonstrated a selective defect in respiratory burst activity but not in degranulation by phagocytic cells in GSD 1b but not in GSD 1a. The respiratory burst abnormality in phagocytic cells from GSD 1b patients was associated with impaired calcium mobilization, whereas these processes were normal in GSD 1a patients. Therefore, the alteration in calcium mobilization was an indication of a signalling defect in phagocytic cells from GSD 1b. However, calcium mobilization was normal in lymphocytes, indicating that defective calcium mobilization was not a global finding in circulating leukocytes, but was specific to phagocytic cells. Calcium mobilization in response to ionomycin was reduced suggesting decreased calcium stores in GSD 1b neutrophils. Therefore, altered phagocytic cell function in GSD 1b patients appears to be associated with diminished calcium mobilization and defective calcium stores. This defective calcium signalling was associated with a selective defect in respiratory burst activity but not degranulation.

Alpha-1-antichymotrypsin inhibits the NADPH oxidase-enzyme complex in phorbol ester-stimulated neutrophil membranes.

The generation of superoxide anion and release of granule contents are essential to the bactericidal function of neutrophils, but may also contribute to host tissue damage during inflammation. In previous studies (J. Immunol. 146:2388), we have demonstrated that the acute phase reactant alpha-1-antichymotrypsin (ACT), a potent inhibitor of the serine protease cathepsin G, also suppresses superoxide anion generation. The inhibitory effect of ACT was not directly linked to its antiproteolytic activity and may reflect interaction at a site other than its reactive loop. To further characterize the mechanism of inhibition, we investigated the direct effects of ACT on the NADPH oxidase enzyme complex and the signaling pathways that regulate motivation of the respiratory burst. We present evidence that ACT does not intefer with agonist-stimulated calcium mobilization or translocation and activity of protein kinase C. ACT was an effective inhibitor of superoxide anion generation in membrane preparations isolated from PMA-activated cells. These results support the notion that ACT is acting on a component of the active assembled NADPH oxidase complex. Thus, ACT may have an important role in regulation of specific aspects of the inflammatory processes and the modulation of toxic oxygen-based host tissue damage.

Acyl-CoA esters modulate intracellular Ca2+ handling by permeabilized clonal pancreatic beta-cells.

Cytosolic free Ca2+ rises in pancreatic beta-cells in response to glucose stimulation and is part of the coupling to insulin secretion. This study evaluates a possible role for cytosolic long chain acyl-CoA esters in modulating Ca2+ handling by clonal beta-cells (HIT). Intact cells incubated with 20 microM free palmitic acid exhibited a 40% decrease in basal cytosolic free Ca2+. In contrast, acyl-CoA esters, up to a chain length of 16, but not the corresponding fatty acids, significantly lowered the Ca2+ set point maintained by cells permeabilized with saponin. The maximum response to the various acyl-CoA esters increased with increasing chain length, with no differences in the half-maximally effective concentration of 0.5 microM. Long chain acyl-CoA esters caused a 40-50% increase in 45Ca2+ influx into a non-mitochondrial pool in the permeabilized HIT cells, consistent with a stimulatory effect on the endoplasmic reticulum Ca(2+)-ATPase activity, but did not affect inositol 1,4,5-trisphosphate-induced Ca(2+)-efflux. Thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+)-ATPase activity, blocked the decrease in the Ca2+ set point caused by acyl-CoA esters. The ability of acyl-CoA esters to lower the Ca2+ set point depended on the ATP/ADP ratio (or free ADP); the Ca2+ set point was lowered by 36 +/- 3.6% at an ATP/ADP ratio of 90 and by 14 +/- 1.9% at an ATP/ADP ratio of 7. Depletion of cellular protein kinase C did not prevent the acyl-CoA-induced lowering of the Ca2+ set point. These findings suggest that the increases in long chain acyl-CoA esters may play a role in restoring cytosolic free Ca2+ through activation of Ca(2+)-ATPases.