Direct Activation Of Protein Kinase Research Articles

Muscarinic Receptor Stimulation Increases Regulators of G-protein Signaling 2 mRNA Levels through a Protein Kinase C-dependent Mechanism

RGS2, a member of the Regulators of G-protein Signaling family, modulates the activity of G-proteins coupled to the phosphoinositide signal transduction system, but little is known about what regulates RGS2. In human neuroblastoma SH-SY5Y cells stimulation of muscarinic receptors by carbachol activates phosphoinositide signaling and also caused a rapid, large, and long lasting increase in RGS2 mRNA levels. Direct activation of protein kinase C also rapidly increased RGS2 mRNA levels. Inhibition of protein kinase C with Ro31-8220, GF109203x, or Go6976 or down-regulation of protein kinase C inhibited increases in RGS2 mRNA levels induced by carbachol or by the activation of protein kinase C. Blockade of calcium signaling did not alter carbachol-induced increases in RGS2 mRNA levels. Neither activation of epidermal growth factor receptors nor stimulation of cyclic AMP production with forskolin increased RGS2 mRNA levels. Pretreatment with actinomycin D blocked increases in RGS2 mRNA levels but caused a surprisingly small, although statistically significant, partial blockade of protein kinase C-mediated feedback inhibition of carbachol-induced phosphoinositide hydrolysis. Thus, RGS2 mRNA levels are increased by activation of muscarinic receptors coupled to the phosphoinositide signal transduction system through a protein kinase C-dependent mechanism. This action may contribute to negative feedback control of this signaling cascade, but because the small contribution to negative feedback contrasts with the large and prolonged elevations in RGS2 mRNA levels, we speculate that its primary role may be in modulating other signaling components.

Low and nontoxic levels of ionic mercury interfere with the regulation of cell growth in the WEHI-231 B-cell lymphoma.

WEHI-231 is a mouse B-cell line, which is a well-established model for studying signal transduction in B lymphocytes, normally responding to cross-linking of the B-cell receptor (BCR) complex by the rapid upregulation of protein tyrosine kinase activity, followed by increased intracellular calcium and activation of protein kinase C. In WEHI-231, activation of protein kinase C is functionally associated with downregulation of DNA synthesis, followed by the induction of apoptosis. We have found in WEHI-231, that at low and environmentally relevant exposure levels (0.1 microM) mercury is not toxic, but still interferes with signal transduction in that it attenuates the growth inhibitory effects of BCR cross-linking. The molecular target for mercury resulting in attenuation of the BCR-mediated growth inhibitory signal is likely proximal to activation of the BCR complex, as HgCl2 had no effect on the negative growth signal generated downstream by direct activation of protein kinase C with phorbol 12-myristate 13-acetate. Treatment of WEHI-231 cells with high and toxic concentrations of Hg results in a marked increase in protein tyrosine phosphorylation in a great many proteins; whereas treatment of WEHI-231 cells with 0.1 microM mercury is not toxic. Under these conditions mercury selectively perturbed BCR-mediated protein tyrosine phosphorylation of a 75 kDa protein, without grossly affecting tyrosine phosphorylation levels of most other proteins. These data suggest that low levels of mercury, which are not toxic, may still contribute to immune dysfunction by interfering with antigen-receptor-mediated and protein-kinase-dependent signal transduction in lymphocytes.

Gβγ-Mediated Regulation of Golgi Organization Is through the Direct Activation of Protein Kinase D

We have shown previously that the βγ subunits of the heterotrimeric G proteins regulate the organization of the pericentriolarly localized Golgi stacks. In this report, evidence is presented that the downstream target of Gβγ is protein kinase D (PKD), an isoform of protein kinase C. PKD, unlike other members of this class of serine/threonine kinases, contains a pleckstrin homology (PH) domain. Our results demonstrate that Gβγ directly activates PKD by interacting with its PH domain. Inhibition of PKD activity through the use of pharmacological agents, synthetic peptide substrates, and, more specifically, the PH domain of PKD prevents Gβγ-mediated Golgi breakdown. Our findings suggest a possible mechanism by which the direct interaction of Gβγ with PKD regulates the dynamics of Golgi membranes and protein secretion.

PAR1 activation initiates integrin engagement and outside-in signalling in megakaryoblastic CHRF-288 cells

To better understand the means by which cells such as human platelets regulate the binding of the integrin α IIbβ 3 to fibrinogen, we have examined agonist-initiated inside-out and outside-in signalling in CHRF-288 cells, a megakaryoblastic cell line that expresses α IIbβ 3 and the human thrombin receptor, PAR1. The results show several notable similarities and differences. (1) Activation of PAR1 caused CHRF-288 cells to adhere and spread on immobilized fibrinogen in an α IIbβ 3-dependent manner, but did not support the binding of soluble fibrinogen or PAC-1, an antibody specific for activated α IIbβ 3. (2) Direct activation of protein kinase C with PMA or disruption of the actin cytoskeleton with low concentrations of cytochalasin D also caused CHRF-288 cells to adhere to fibrinogen. (3) Despite the failure to bind soluble fibrinogen, activation of PAR1 in CHRF-288 cells caused phosphoinositide hydrolysis, arachidonate mobilization and the phosphorylation of p42 MAPK, phospholipase A 2 and the Rac exchange protein, Vav, all of which occur in platelets. PAR1 activation also caused an increase in cytosolic Ca 2+, which, when prevented, blocked adhesion to fibrinogen. (4) Finally, as in platelets, adhesion of CHRF-288 cells to fibrinogen was followed by a burst of integrin-dependent (‘outside-in’) signalling, marked by FAK phosphorylation and a more prolonged phosphorylation of p42 MAPK. However, in contrast to platelets, adhesion to fibrinogen had no effect on Vav phosphorylation. Collectively, these observations show that signalling initiated through PAR1 in CHRF-288 cells can support α IIbβ 3 binding to immobilized ligand, but not the full integrin activation needed to bind soluble ligand. This would suggest that there has been an increase in integrin avidity without an accompanying increase in affinity. Such increases in avidity are thought to be due to integrin clustering, which would also explain the results obtained with cytochalasin D. The failure of α IIbβ 3 to achieve the high affinity state in CHRF-288 cells was not due to the failure of PAR1 activation to initiate a number of signalling events that normally accompany platelet activation nor did it prevent at least some forms of outside-in signalling. However, at least one marker of outside-in signalling, the augmentation of Vav phosphorylation seen during platelet aggregation, did not occur in CHRF-288 cells.

M3 Muscarinic Acetylcholine Receptors Regulate Cytoplasmic Myosin by a Process Involving RhoA and Requiring Conventional Protein Kinase C Isoforms

Although muscarinic acetylcholine receptors (mAChR) regulate the activity of smooth muscle myosin, the effects of mAChR activation on cytoplasmic myosin have not been characterized. We found that activation of transfected human M3 mAChR induces the phosphorylation of myosin light chains (MLC) and the formation of myosin-containing stress fibers in Chinese hamster ovary (CHO-m3) cells. Direct activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) also induces myosin light chain phosphorylation and myosin reorganization in CHO-m3 cells. Conventional (alpha), novel (delta), and atypical (iota) PKC isoforms are activated by mAChR stimulation or PMA treatment in CHO-m3 cells, as indicated by PKC translocation or degradation. mAChR-mediated myosin reorganization is abolished by inhibiting conventional PKC isoforms with Go6976 (IC50 = 0.4 microM), calphostin C (IC50 = 2.4 microM), or chelerythrine (IC50 = 8.0 microM). Stable expression of dominant negative RhoAAsn-19 diminishes, but does not abolish, mAChR-mediated myosin reorganization in the CHO-m3 cells. Similarly, mAChR-mediated myosin reorganization is diminished, but not abolished, in CHO-m3 cells which are multi-nucleate due to inactivation of Rho with C3 exoenzyme. Expression of dominant negative RhoAAsn-19 or inactivation of RhoA with C3 exoenzyme does not affect PMA-induced myosin reorganization. These findings indicate that the PKC-mediated pathway of myosin reorganization (induced either by M3 mAChR activation or PMA treatment) can continue to operate even when RhoA activity is diminished in CHO-m3 cells. Conventional PKC isoforms and RhoA may participate in separate but parallel pathways induced by M3 mAChR activation to regulate cytoplasmic myosin. Changes in cytoplasmic myosin elicited by M3 mAChR activation may contribute to the unique ability of these receptors to regulate cell morphology, adhesion, and proliferation.

Interplay between the NO pathway and elevated [Ca2+]i enhances ciliary activity in rabbit trachea.

1. Average intracellular calcium concentration ([Ca2+]i) and ciliary beat frequency (CBF) were simultaneously measured in rabbit airway ciliated cells in order to elucidate the molecular events that lead to ciliary activation by purinergic stimulation. 2. Extracellular ATP and extracellular UTP caused a rapid increase in both [Ca2+]i and CBF. These effects were practically abolished by a phospholipase C inhibitor (U-73122) or by suramin. 3. The effects of extracellular ATP were not altered: when protein kinase C (PKC) was inhibited by either GF 109203X or chelerythrine chloride, or when protein kinase A (PKA) was inhibited by RP-adenosine 3', 5'-cyclic monophosphothioate triethylamine (Rp-cAMPS). 4. Activation of PKC by phorbol 12-myristate, 13-acetate (TPA) had little effect on CBF or on [Ca2+]i, while activation of PKA by forskolin or by dibutyryl-cAMP led to a small rise in CBF without affecting [Ca2+]i. 5. Direct activation of protein kinase G (PKG) with dibutyryl-cGMP had a negligible effect on CBF when [Ca2+]i was at basal level. However, dibutyryl-cGMP strongly elevated CBF when [Ca2+]i was elevated either by extracellular ATP or by ionomycin. 6. The findings suggest that the initial rise in [Ca2+]i induced by extracellular ATP activates the NO pathway, thus leading to PKG activation. In the continuous presence of elevated [Ca2+]i the stimulated PKG then induces a robust enhancement in CBF. In parallel, activated PKG plays a central role in Ca2+ influx via a still unidentified mechanism, and thus, through positive feedback, maintains CBF close to its maximal level in the continuous presence of ATP.

Nitric Oxide Donors Induce Stress Signaling via Ceramide Formation in Rat Renal Mesangial Cells

Exogenous NO is able to trigger apoptosis of renal mesangial cells, and thus may contribute to acute lytic phases as well as to resolution of glomerulonephritis. However, the mechanism involved in these events is still unclear. We report here that chronic exposure of renal mesangial cells for 24 h to compounds releasing NO, including spermine-NO, (Z)-1-{N-methyl-N-[6-(N-methylammoniohexyl)amino]}diazen-1-ium-1, 2-diolate (MAHMA-NO), S-nitrosoglutathione (GS-NO), and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) results in a potent and dose-dependent increase in the lipid signaling molecule ceramide. Time courses reveal that significant effects occur after 2-4 h of stimulation with NO donors and reach maximal levels after 24 h of stimulation. No acute (within minutes) ceramide production can be detected. When cells were stimulated with NO donors in the presence of phorbol ester, a direct activator of protein kinase C, both ceramide production and DNA fragmentation are completely abolished. Furthermore, addition of exogenous ceramide partially reversed the inhibitory effect of phorbol ester on apoptosis, thus suggesting a negative regulation of protein kinase C on ceramide formation and apoptosis. In contrast to exogenous NO, tumor necrosis factor (TNF)-alpha stimulates a very rapid and transient increase in ceramide levels within minutes but fails to induce the late-phase ceramide formation. Moreover, TNF fails to induce apoptosis in mesangial cells. Interestingly, NO and TNFalpha cause a chronic activation of acidic and neutral sphingomyelinases, the ceramide-generating enzymes, whereas acidic and neutral ceramidases, the ceramide-metabolizing enzymes, are inhibited by NO, but potently stimulated by TNFalpha. Furthermore, in the presence of an acidic ceramidase inhibitor, N-oleoylethanolamine, TNFalpha leads to a sustained accumulation of ceramide and in parallel induces DNA fragmentation. In summary, our data demonstrate that exogenous NO causes a chronic up-regulation of ceramide levels in mesangial cells by activating sphingomyelinases and concomitantly inhibiting ceramidases, and that particularly the late-phase of ceramide generation may be responsible for the further processing of a proapoptotic signal.

Measurement of inositol (poly)phosphate formation using [3H]inositol labeling protocols in permeabilized cells.

AbstractHormones, neurotransmitters, chemoattractants, and growth factors all elicit intracellular responses, on binding to cell surface receptors, by activating inositol phospholipid-specific phospholipase C (PLC). Activated PLC catalyzes the hydrolysis of phosphatidylinositol bisphosphate (PIP2), a minor membrane phospholipid, to form two second messengers, diacylglycerol (DAG) and inositol (1,4,5)trisphosphate [Ins(1,4,5,)P3]. DAG is a direct activator of protein kinase C isozymes, Ins(1,4,5)P3 mobilizes intracellular Ca2+. G protein-coupled receptors couple to the PLC-β family via G proteins, and tyrosine kinase receptors activate PLCγ isozymes (1). Regardless of the PLC isozyme activated, the product is invariantly Ins(1,4,5)P3.KeywordsHL60 CellPermeabilized CellInositol PhosphateInositol TrisphosphateAssay TubeThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Increased NAD(P)H oxidase-mediated superoxide production in renovascular hypertension: Evidence for an involvement of protein kinase C

Angiotensin II infusion has been shown to cause hypertension and endothelial dysfunction and to increase superoxide (O-.2) production in vascular tissue, mainly via an activation of nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]-dependent oxidase, the most significant O-.2 source in endothelial and/or smooth muscle cells. With these studies, we sought to determine whether endothelial dysfunction in renovascular hypertension is secondary to an activation of these oxidases. Endothelial function in aortas from rats with two kidney-one clip (2K-1C) hypertension and age-matched controls was assessed using isometric tension studies in organ chambers. Changes in vascular O-.2 production were measured using lucigenin-enhanced chemiluminescence and electron spin resonance spectroscopy. In hypertensive animals, relaxation to endothelium-dependent (acetylcholine) and endothelium-independent nitrovasodilators (nitroglycerin) was impaired. Constriction to a direct activator of protein kinase C (PKC) phorbol ester 12,13 dibutyrate (PDBu) was enhanced, and vascular O-.2 was significantly increased compared with controls. Vascular O-.2 was normalized by the PKC inhibitor calphostin C, by the inhibitor of flavin-dependent oxidases, diphenylene iodonium, and recombinant heparin-binding superoxide dismutase, whereas inhibitors of the xanthine oxidase (oxypurinol), nitric oxide synthase (NG-nitro-l-arginine) and mitochondrial NADH dehydrogenase (rotenone) were ineffective. Studies of vascular homogenates demonstrated that the major source of O-.2 was a NAD(P)H-dependent oxidase. Incubation of intact tissue with PDBu markedly increased O-. 2, the increase being significantly stronger in vessels from hypertensive animals as compared with vessels from controls. Endothelial dysfunction was improved by preincubation of vascular tissue with superoxide dismutase and calphostin C. We therefore conclude that renovascular hypertension in 2K-1C rats is associated with increased vascular O-.2 leading to impaired vasodilator responses to endogenous and exogenous nitrovasodilators. Increased vascular O-.2 is likely secondary to a PKC-mediated activation of a membrane-associated NAD(P)H-dependent oxidase.

Sulfonylureas enhance exocytosis from pancreatic beta-cells by a mechanism that does not involve direct activation of protein kinase C.

Hypoglycemic sulfonylureas stimulate insulin release by binding to a regulatory subunit of plasma membrane ATP-sensitive K+ (K(ATP)) channels. The consequent closure of K(ATP) channels leads to depolarization, opening of voltage-dependent Ca2+ channels, Ca2+ influx, and a rise in intracellular [Ca2+]. Recently, however, it has been suggested that sulfonylureas may have an additional action on secretion, independent of changes in intracellular [Ca2+] but dependent on the activity of protein kinase C (PKC). We have investigated the mechanisms involved in the PKC-dependent effect of sulfonylureas on the secretion machinery in beta-cells. In MIN6 beta-cells permeabilized by streptolysin O, insulin release was stimulated by elevation of [Ca2+] from 10(-8) to 10(-5) mol/l. At a [Ca2+] of 10(-8) mol/l, insulin release from permeabilized beta-cells was stimulated by addition of GTP-gamma-S, or by addition of a phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA). TPA, but not GTP-gamma-S, also increased insulin release when [Ca2+] was 10(-5) mol/l. Insulin release from permeabilized beta-cells was stimulated by tolbutamide (0.1-1 mmol/l) at 10(-8) but not at 10(-5) mol/l Ca2+. The effect of tolbutamide was blocked either by inhibition of PKC or when phorbol ester-sensitive PKC isoforms were maximally stimulated by TPA. Meglitinide and glibenclamide also stimulated insulin release from permeabilized beta-cells. To assess the possibility that direct activation of PKC mediates the exocytotic response to sulfonylureas, we studied the effect of tolbutamide and glibenclamide on PKC activity. Purified brain PKC was not activated by tolbutamide or glibenclamide, whether tested in the absence or presence of phosphatidylserine or TPA, or at low or high [Ca2+]; nor was the total PKC activity in extracts of MIN6 beta-cells affected by tolbutamide. Neither tolbutamide nor glibenclamide elicited translocation of any isoform of PKC in intact or permeabilized beta-cells under conditions in which TPA evoked a marked redistribution of PKC alpha- and epsilon-isoforms. We conclude that although the plasma membrane K(ATP) channel-independent stimulation of exocytosis by sulfonylureas may require functional PKC, the mechanism does not involve a direct activation of the enzyme.

Protein Kinase C: Modulation of Vasopressin-Induced Calcium Influx and Release in A7r5 Vascular Smooth Muscle Cells

This study was guided by the hypothesis that specific isoforms of protein kinase C may participate in modulating increases in intracellular Ca2+that are induced by stimulation of vascular smooth muscle cells with vasopressin. Immunoblot analysis revealed that A7r5 vascular smooth muscle cells expressed conventional (α), novel (δ and ϵ), and atypical (ι/λ and μ) isoforms of protein kinase C. Stimulation of fura-2-loaded cells with 20 nM vasopressin induced a rapid transient increase in the intracellular concentration of calcium that was followed by a slowly declining component which was above baseline throughout the period of observation. Cell fractionation studies showed that the calcium response was associated with (a) transient translocation of the α and δ isoforms of protein kinase C from the cytosolic fraction to the particulate-membrane fraction, (b) sustained translocation of the ϵ isoform, and (c) no translocation of ι/λ or μ isoforms. Ratiometric and isobestic fluorescence analysis showed that vasopressin-induced Ca2+influx and release were markedly inhibited in cells that were preincubated with either 1 μM phorbol 12-myristate 13-acetate, or 10 μM 1,2 dioctanoyl-sn-glycerol, two structurally different activators of protein kinase C. In contrast, vasopressin-induced increases in intracellular Ca2+were not significantly altered following preincubation with either 1 μM 4α-phorbol or 4α-phorbol 12,13-didecanoate, analogs of phorbol 12-myristate 13-acetate that do not activate protein kinase C. Moreover, the inhibitory effects of phorbol 12-myristate 13-acetate were prevented by treatment with 1 μM GF109203X, a potent inhibitor of protein kinase C. Taken together, these results show that direct activation of protein kinase C can negatively modulate vasopressin-induced Ca2+influx and release in cultured vascular smooth muscle cells. They also show that stimulation with vasopressin induces translocation of specific isoforms of protein kinase C, an observation suggesting that one or more of these isoforms may participate in modulation of vasopressin-induced increases in intracellular Ca2+.

Nitrous oxide impairs the signaling of neutrophils downstream of receptors

(1) Nitrous oxide has been shown to impair the oxidative function of neutrophils. (2) To characterize the type and the site of the drug interaction, receptor expression, cytosolic-free calcium, and H 2O 2 production of neutrophils were assessed using flow cytometry. (3) Nitrous oxide depresses receptor-dependent generation of H 2O 2 in a concentration-dependent manner. The response upon direct activation of protein kinase C (PKC) was unaffected. (4) No interference was found at the receptor sites, the linked G proteins, and the subsequent release of Ca 2+, indicating a localization of the nitrous oxide interaction downstream of receptors and G proteins at or near to PKC.

Platelet Agonists Enhance the Import of Phosphatidylethanolamine into Human Platelets

It is unknown whether the endocytosis-independent transfer of phospholipids from lipoproteins to platelets is regulated by platelet agonists such as thrombin. The movements of the choline phospholipids phosphatidylcholine and sphingomyelin (labeled with either 14C or the fluorescent pyrenedecanoic acid) between low density lipoproteins and platelets were unaffected by thrombin (0.5 unit/ml). In contrast, thrombin accelerated the import of diacyl phosphatidylethanolamine (PE) and alkenylacyl phosphatidylethanolamine into platelets by about 4-fold. Similarly, thrombin receptor-activating peptide (15 microM), collagen (10 microgram/ml), and ADP (10 microM) enhanced PE uptake. High density lipoprotein particles and egg phosphatidylcholine vesicles were also donors for stimulation of platelet PE import. Part of the [14C]arachidonic acid-labeled PE transferred from low density lipoprotein to platelets activated by thrombin and collagen was metabolized to 14C-eicosanoids. Inhibitors of protein kinase C partially prevented thrombin-induced [14C]PE uptake, while direct activators of protein kinase C increased incorporation of [14C]PE into platelets. Proteinaceous factor(s) recovered in the extracellular medium from ADP- and thrombin-activated platelet suspensions were found to accelerate the transfer of pyrenedecanoic acid-labeled PE between donor and acceptor lipid vesicles. The stimulation of import of ethanolamine phospholipids led to a 2-fold enhancement of the prothrombinase activity of thrombin-activated platelets. Our study demonstrates that physiological platelet stimuli increase specifically the transfer of ethanolamine phospholipids from lipoproteins to platelets through a secretion-dependent mechanism. This might contribute to the increase of procoagulant activity of stimulated platelets.

Antimitogenic effects of trapidil in coronary artery smooth muscle cells by direct activation of protein kinase A.

The triazolopyrimidine trapidil has been found in controlled clinical trials to prevent restenosis after vascular injury. Although trapidil is widely regarded as a platelet-derived growth factor receptor (PDGF) antagonist, its precise mode of action is still unknown. This study was designed to investigate the inhibition of mitogenesis by trapidil in cultured bovine coronary artery smooth muscle cells (SMC) and to identify major signal transduction pathways involved. Trapidil inhibited PDGF-BB-induced mitogenesis in SMC in a concentration-dependent manner. Comparable inhibitory effects were obtained after stimulation of smooth muscle cells by phorbol ester, which suggests that the action of trapidil was not restricted to PDGF receptor-mediated mechanisms. Trapidil also inhibited PDGF- and phorbol ester-induced mitogen-activated protein kinase as well as Raf-1 kinase activity. As a possible target of trapidil, stimulation of cellular protein kinase A (PKA) activity was detected. Trapidil also induced the phosphorylation of vasodilator-stimulated phosphoprotein in SMC. Antimitogenic effects of trapidil were completely abolished by PKA inhibitors. Neither a direct stimulation of cAMP formation nor a phosphodiesterase inhibition was observed at antimitogenic concentrations of trapidil. However, trapidil directly activated purified PKA holoenzyme in a cAMP-independent manner. In conclusion, trapidil exerts its antimitogenic effects on SMC by direct activation of PKA. Thus, PKA-mediated inhibition of the Raf-1/MAP kinase pathway may be involved in the antimitogenic actions of the compound.

Protein kinase C isoforms in normal and chronic myeloid leukemic neutrophils. Distinct signal for PKC α by immunodetection on PVDF membrane, decreased expression of PKC α and increased expression of PKC δ in leukemic neutrophils

Neutrophils from patients with Chronic Myeloid Leukemia (CML) exhibit defects in several functions. They also show altered phosphorylation–dephosphorylation patterns of several proteins on stimulation with phorbol myristate acetate—a direct activator of protein kinase C (PKC). Since PKC mediates several functions of the neutrophil, in this study we investigate the PKC isoform profile and subcellular distribution in normal and CML neutrophils in an attempt to elucidate their role in CML signalling. Our results show the presence of PKC α, βI, βII and δ in both the cell types. A distinct and clear signal was obtained for PKC α, the isoform reported to be absent or present in very low amounts in normal neutrophils. In addition, PKC α was present in significantly lower levels in CML neutrophils while the PKC δ isoform was found in significantly higher amounts in the CML cytosol as compared to that in normal cells. PKC α, βI, βII and δ isoforms could not be detected in the nucleus of unstimulated normal and CML neutrophils. The altered levels of PKC α and δ may be one of the causes for the defects in function exhibited by the leukemic cells.

Suppressed PHA activation of T lymphocytes in simulated microgravity is restored by direct activation of protein kinase C.

Utilizing clinostatic rotating wall vessel (RWV) bioreactors that simulate aspects of microgravity, we found phytohemagglutinin (PHA) responsiveness to be almost completely diminished. Activation marker expression was significantly reduced in RWV cultures. Furthermore, cytokine secretion profiles suggested that monocytes are not as adversely affected by simulated microgravity as T cells. Reduced cell-cell and cell-substratum interactions may play a role in the loss of PHA responsiveness because placing peripheral blood mononuclear cells (PBMC) within small collagen beads did partially restore PHA responsiveness. However, activation of purified T cells with cross-linked CD2/CD28 and CD3/CD28 antibody pairs was completely suppressed in the RWV, suggesting a defect in signal transduction. Activation of purified T cells with PMA and ionomycin was unaffected by RWV culture. Furthermore, sub-mitogenic doses of PMA alone but not ionomycin alone restored PHA responsiveness of PBMC in RWV culture. Thus our data indicate that during polyclonal activation the signaling pathways upstream of PKC activation are sensitive to simulated microgravity.

Nitrous oxide impairs the neutrophil oxidative response.

Nitrous oxide has been shown inconsistently to impair the oxidative function of neutrophils. The choice of the stimulus, receptor agonists, or stimuli acting independent of receptors seems to determine whether nitrous oxide impairs the oxidative functions, suggesting an interference with the cytosolic signaling of neutrophils. Production of hydrogen peroxide by neutrophils was assessed using flow cytometric analysis. N-formyl-methionyl-leucyl-phenylalanine (FMLP), C5a, dioctanylglycerol, and phorbol-12-myristate-13-acetate were used as stimuli. In addition, the expression of receptors for FMLP and the cytosolic-free calcium response of cells were measured. Nitrous oxide depresses C5a- or FMLP-induced generation of reactive oxygen derivatives in a concentration-dependent manner. However, the response with direct activation of protein kinase C was unaffected. Further, the number of FMLP receptors and the cytosolic calcium response were unaffected. Inhibition of the oxidative response was not reversible within the observation period of 4 h. Nitrous oxide inhibited the intracellular signaling of the investigated G-protein-coupled receptors for chemotactic peptides. No interference of nitrous oxide with reduced nicotinamide adenine dinucleotide phosphate oxidase, the oxidative enzyme system of neutrophils, nor with its activation through protein kinase C was detected.

Regulation of 10P2 murine mast cell proliferation and secretory function by stem cell factor or IL-9.

Mast cells are effectors of inflammatory responses. When triggered by immunological or nonimmunological mechanisms, mast cells release potent biological mediators from preformed stores and synthesize others de novo. In previous investigations from this laboratory, the signal transduction pathways of cloned 10P2 cytokine-independent mast cells were explored. Results suggested that 10P2 cells undergo activation-secretion coupling assessed as release of stored [14C]serotonin (5-HT) when challenged with IgE-specific antigen, influx of extracellular calcium, release of intracellular calcium stores, or by direct activation of protein kinase C isozymes. In the present investigations, cytokine proliferative effects and modulatory roles on release of stored [14C]5-HT have been explored. Following passive sensitization with anti-dinitrophenol (anti-DNP) IgE and challenge with DNP, mast cells released up to 32% of the stored [14C]5-HT. Pretreatment of cells with 10, 30, or 50 ng/ml stem cell factor (SCF) did not alter the response. SCF did not directly induce [14C]5-HT release. Pretreatment with 25 ng/ml interleukin-9 (IL-9) significantly potentiated the IgE-antigen release by 51.1%, 35.7%, or 31.6% when challenged with 3, 10 or 30 ng/ml DNP-HSA. Treatment of cells with 1-100 ng/ml SCF for 72 hr resulted in significantly enhanced proliferation whereas this did not occur when cells were treated with 1-100 ng/ml IL-9. Collectively, these results suggest that SCF alone has a proliferative effect, does not alter the IgE-specific antigen signal transduction pathway, and does not directly stimulate mast cell degranulation. In contrast, IL-9 potentiates the IgE-antigen signal transduction response but exerts no proliferative response. Reports of effects of orally administered cytokines are now beginning to emerge. This raises the possibility that cytokines may be a future therapeutic approach to treatment of allergic and nonallergic inflammatory diseases. The 10P2 cytokine-independent mast cell line may be a valuable adjunct to existing mast cell models as this avenue of drug discovery is explored.

Characterization of Early Activation Events in Cord Blood B Cells after Stimulation with T Cell-Independent Activators

Human neonates are immunologically immature, particularly in their humoral antibody responses to T cell-independent antigens, as exemplified by their increased susceptibility to infections with polysaccharide-encapsulated bacteria. To clarify the mechanism(s) underlying the unresponsiveness of neonates to polysaccharide antigens, we used an in vitro model with neonatal cord blood cells that has been shown to mimic surface Ig-dependent signaling in the adult by T cell-independent antigens. We studied the ability of cord blood human B cells to become activated after ligation of their surface Ig by unconjugated anti-Ig, dextran-conjugated anti-Ig, and Staphylococcus aureus Cowan A1, and compared their response with that of adult B cells. After the addition of nanogram concentrations of anti-Ig-dextran, neonatal cord blood B cells proliferated at levels comparable to that observed with adult B cells. The majority of cord blood B cells showed a marked rise in intracellular calcium, increased surface expression of human leukocyte antigen DR, and an increase in cell size. Direct activation of protein kinase C by phorbol esters in neonatal B cells led to cellular proliferation, and when combined with anti-Ig, a synergistic effect on proliferation was observed. These data suggest that the unresponsiveness of human neonates to polysaccharide antigens does not represent an inability of these antigens to induce early activation events in circulating B cells.

Specific Inhibition of Phorbol Ester-stimulated Phospholipase D by Clostridium sordellii Lethal Toxin and Clostridium difficile Toxin B-1470 in HEK-293 Cells: RESTORATION BY Ral GTPases

Activation of m3 muscarinic acetylcholine receptor (mAChR), stably expressed in human embryonic kidney (HEK)-293 cells, leads to phospholipase D (PLD) stimulation, a process apparently involving Rho GTPases, as shown by studies with Clostridium botulinum C3 exoenzyme and Clostridium difficile toxin B (TcdB). Direct activation of protein kinase C (PKC) by phorbol esters, such as phorbol 12-myristate 13-acetate (PMA), also induces PLD stimulation, which is additive to the mAChR action and which is only poorly sensitive to inactivation of Rho proteins by TcdB. To study whether Ras-like GTPases are involved in PLD regulation, we studied the effects of the TcdB variant TcdB-1470 and Clostridium sordellii lethal toxin (TcsL), known to inactivate Rac and some members of the Ras protein family, on PLD activities. TcdB-1470 and TcsL did not affect basal PLD activity and PLD stimulation by mAChR or direct G protein activation. In contrast, PMA-induced PLD stimulation was inhibited by TcdB-1470 and TcsL in a time- and concentration-dependent manner, without alteration in immunologically detectable PKC isozyme levels. In membranes of HEK-293 cells pretreated with TcdB-1470 or TcsL, basal and stable GTP analog-stimulated PLD activities measured with exogenous phosphatidylcholine, in the presence or absence of phosphatidylinositol 4,5-bisphosphate, were not altered. In contrast, pretreatment with TcdB-1470 and TcsL, but not TcdB, strongly reduced PMA-stimulated PLD activity. The addition of recombinant Rac1, serving as glucosylation substrate for TcdB, TcsL, and TcdB-1470, did not restore PLD stimulation by PMA. Furthermore, PMA-stimulated PLD activity, suppressed by prior treatment with TcdB-1470 or TcsL, was not rescued by the addition of recombinant Ras (RasG12V) or Rap proteins, acting as glucosylation substrates for TcsL only (Ras) or TcdB-1470 and TcsL (Rap). In contrast, the addition of recombinant Ral proteins (RalA and RalB), glucosylation substrates for TscL and TcdB-1470, but not for TcdB, to membranes of TcdB-1470- or TcsL-treated cells fully restored PLD stimulation by PMA without altering the strict MgATP dependence of PMA-induced PLD stimulation. RalA-mediated restoration of PMA-stimulated PLD activity in membranes of TcsL-treated cells was not enhanced by coaddition of RasG12V. In conclusion, the data presented indicate that TcdB-1470 and TcsL selectively interfere with phorbol ester stimulation of PLD and suggest an essential role of Ral proteins in PKC signaling to PLD in HEK-293 cells.