Effects of asbestos and silica on superoxide anion production in the guinea pig alveolar macrophage

Asbestos and silica are important industrial hazards. Exposure to these dusts can result in pulmonary fibrosis and, in the case of asbestos, cancer. Although the hazards of asbestos and silica exposure have long been known, the pathogenesis of dust-related disease is not well understood. Both silica and asbestos are thought to alter the function of the alveolar macrophage, but the nature of the biochemical alteration is unknown. Therefore, this study examined the effect of asbestos and silica on the activation pathway of the guinea pig alveolar macrophage. Activation of macrophages by physiological agents results in stimulation of phospholipase C causing phosphatidyl inositol turnover and intracellular calcium mobilization. Phosphatidyl inositol turnover produces diacylglycerol which activates protein kinase C causing superoxide anion production.^ Chrysotile stimulated alveolar macrophages to produce superoxide anion. This stimulation proceeded via phospholipase C, since chrysotile stimulated phosphatidyl inositol turnover and intracellular calcium mobilization. The possible involvement of a coupling protein was evaluated by pretreating cells with pertussis toxin. Pertussis toxin pretreatment partially inhibited chrysotile stimulation, suggesting that chrysotile activates a coupling protein in an non-classical manner. Potential binding sites for chrysotile stimulation were examined using a series of nine lectins. Chrysotile-stimulated superoxide anion production was blocked by pretreatment with lectins which bound to N-acetylglucosamine, but not by lectins which bound to mannose, fucose, or N-acetylgalactosamine. In addition, incubation with the N-acetylglucosamine polymer, chitin, inhibited chrysotile-stimulated superoxide anion production, suggesting that chrysotile stimulated superoxide anion production by binding to N-acetylglucosamine residues.^ On the other hand, silica did not stimulate superoxide anion production. The effect of silica on agonist stimulation of this pathway was examined using two stimulants of superoxide anion production, N-formyl-nle-leu-phe (FNLP, which stimulates through phospholipase C) and phorbol-12,13-dibutyrate (which directly activates protein kinase C). Sublethal doses of silica inhibited FNLP-stimulated superoxide anion production, but did not affect phorbol-12,13-dibutyrate-stimulated superoxide anion production, suggesting that the site of inhibition precedes protein kinase C. This inhibition was not due to cell membrane damage, since cell permeability to calcium-45 and rubidium-86 was not increased. It is concluded that chrysotile binds to N-acetylglucosamine residues on macrophage surface glycoproteins to stimulate the physiological pathway resulting in superoxide anion production. In contrast, silica does not stimulate superoxide anion production, but it did inhibit FNLP-stimulated superoxide anion production. ^

Effects of increased CO2 on fish gill and plasma proteome

Ocean acidification and warming are both primarily caused by increased levels of atmospheric CO2, and marine organisms are exposed to these two stressors simultaneously. Although the effects of temperature on fish have been investigated over the last century, the long-term effects of moderate CO2 exposure and the combination of both stressors are almost entirely unknown. A proteomics approach was used to assess the adverse physiological and biochemical changes that may occur from the exposure to these two environmental stressors. We analysed gills and blood plasma of Atlantic halibut (Hippoglossus hippoglossus) exposed to temperatures of 12°C (control) and 18°C (impaired growth) in combination with control (400 µatm) or high-CO2 water (1000 µatm) for 14 weeks. The proteomic analysis was performed using two-dimensional gel electrophoresis (2DE) followed by Nanoflow LC-MS/MS using a LTQ-Orbitrap. The high-CO2 treatment induced the up-regulation of immune system-related proteins, as indicated by the up-regulation of the plasma proteins complement component C3 and fibrinogen beta chain precursor in both temperature treatments. Changes in gill proteome in the high-CO2 (18°C) group were mostly related to increased energy metabolism proteins (ATP synthase, malate dehydrogenase, malate dehydrogenase thermostable, and fructose-1,6-bisphosphate aldolase), possibly coupled to a higher energy demand. Gills from fish exposed to high-CO2 at both temperature treatments showed changes in proteins associated with increased cellular turnover and apoptosis signalling (annexin 5, eukaryotic translation elongation factor 1 gamma, receptor for protein kinase C, and putative ribosomal protein S27). This study indicates that moderate CO2-driven acidification, alone and combined with high temperature, can elicit biochemical changes that may affect fish health.

Expression of the peroxisome proliferator-activated receptor γ (PPARγ) in human atherosclerosis and regulation in macrophages by colony stimulating factors and oxidized low density lipoprotein

The peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-dependent transcription factor that has been demonstrated to regulate fat cell development and glucose homeostasis. PPARγ is also expressed in a subset of macrophages and negatively regulates the expression of several proinflammatory genes in response to natural and synthetic ligands. We here demonstrate that PPARγ is expressed in macrophage foam cells of human atherosclerotic lesions, in a pattern that is highly correlated with that of oxidation-specific epitopes. Oxidized low density lipoprotein (oxLDL) and macrophage colony-stimulating factor, which are known to be present in atherosclerotic lesions, stimulated PPARγ expression in primary macrophages and monocytic cell lines. PPARγ mRNA expression was also induced in primary macrophages and THP-1 monocytic leukemia cells by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). Inhibition of protein kinase C blocked the induction of PPARγ expression by TPA, but not by oxLDL, suggesting that more than one signaling pathway regulates PPARγ expression in macrophages. TPA induced the expression of PPARγ in RAW 264.7 macrophages by increasing transcription from the PPARγ1 and PPARγ3 promoters. In concert, these observations provide insights into the regulation of PPARγ expression in activated macrophages and raise the possibility that PPARγ ligands may influence the progression of atherosclerosis.

Requirement of GM2 ganglioside activator for phospholipase D activation

Sequence analysis of a heat-stable protein necessary for the activation of ADP ribosylation factor-dependent phospholipase D (PLD) reveals that this protein has a structure highly homologous to the previously known GM2 ganglioside activator whose deficiency results in the AB-variant of GM2 gangliosidosis. The heat-stable activator protein indeed has the capacity to enhance enzymatic conversion of GM2 to GM3 ganglioside that is catalyzed by β-hexosaminidase A. Inversely, GM2 ganglioside activator purified separately from tissues as described earlier [Conzelmann, E. & Sandhoff, K. (1987) Methods Enzymol. 138, 792–815] stimulates ADP ribosylation factor-dependent PLD in a dose-dependent manner. At higher concentrations of ammonium sulfate, the PLD activator protein apparently substitutes for protein kinase C and phosphatidylinositol 4,5-bisphosphate, both of which are known as effective stimulators of the PLD reaction. The mechanism of action of the heat-stable PLD activator protein remains unknown.

Isolation of a multispecific organic anion and cardiac glycoside transporter from rat brain

A novel multispecific organic anion transporting polypeptide (oatp2) has been isolated from rat brain. The cloned cDNA contains 3,640 bp. The coding region extends over 1,983 nucleotides, thus encoding a polypeptide of 661 amino acids. Oatp2 is homologous to other members of the oatp gene family of membrane transporters with 12 predicted transmembrane domains, five potential glycosylation, and six potential protein kinase C phosphorylation sites. In functional expression studies in Xenopus laevis oocytes, oatp2 mediated uptake of the bile acids taurocholate (Km ≈ 35 μM) and cholate (Km ≈ 46 μM), the estrogen conjugates 17β-estradiol-glucuronide (Km ≈ 3 μM) and estrone-3-sulfate (Km ≈ 11 μM), and the cardiac gylcosides ouabain (Km ≈ 470 μM) and digoxin (Km ≈ 0.24 μM). Although most of the tested compounds are common substrates of several oatp-related transporters, high-affinity uptake of digoxin is a unique feature of the newly cloned oatp2. On the basis of Northern blot analysis under high-stringency conditions, oatp2 is highly expressed in brain, liver, and kidney but not in heart, spleen, lung, skeletal muscle, and testes. These results provide further support for the overall significance of oatps as a new family of multispecific organic anion transporters. They indicate that oatp2 may play an especially important role in the brain accumulation and toxicity of digoxin and in the hepatobiliary and renal excretion of cardiac glycosides from the body.

The synthesis of ATP by glycolytic enzymes in the postsynaptic density and the effect of endogenously generated nitric oxide

The major contribution of this paper is the finding of a glycolytic source of ATP in the isolated postsynaptic density (PSD). The enzymes involved in the generation of ATP are glyceraldehyde-3-phosphate dehydrogenase (G3PD) and phosphoglycerate kinase (PGK). Lactate dehydrogenase (LDH) is available for the regeneration of NAD+, as well as aldolase for the regeneration of glyceraldehyde-3-phosphate (G3P). The ATP was shown to be used by the PSD Ca2+/calmodulin-dependent protein kinase and can probably be used by two other PSD kinases, protein kinase A and protein kinase C. We confirmed by immunocytochemistry the presence of G3PD in the PSD and its binding to actin. Also present in the PSD is NO synthase, the source of NO. NO increases the binding of NAD, a G3PD cofactor, to G3PD and inhibits its activity as also found by others. The increased NAD binding resulted in an increase in G3PD binding to actin. We confirmed the autophosphorylation of G3PD by ATP, and further found that this procedure also increased the binding of G3PD to actin. ATP and NO are connected in that the formation of NO from NOS at the PSD resulted, in the presence of NAD, in a decrease of ATP formation in the PSD. In the discussion, we raise the possible roles of G3PD and of ATP in protein synthesis at the PSD, the regulation by NO, as well as the overall regulatory role of the PSD complex in synaptic transmission.

Association of INAD with NORPA is essential for controlled activation and deactivation of Drosophila phototransduction in vivo

Visual transduction in Drosophila is a G protein-coupled phospholipase C-mediated process that leads to depolarization via activation of the transient receptor potential (TRP) calcium channel. Inactivation-no-afterpotential D (INAD) is an adaptor protein containing PDZ domains known to interact with TRP. Immunoprecipitation studies indicate that INAD also binds to eye-specific protein kinase C and the phospholipase C, no-receptor-potential A (NORPA). By overlay assay and site-directed mutagenesis we have defined the essential elements of the NORPA–INAD association and identified three critical residues in the C-terminal tail of NORPA that are required for the interaction. These residues, Phe-Cys-Ala, constitute a novel binding motif distinct from the sequences recognized by the PDZ domain in INAD. To evaluate the functional significance of the INAD–NORPA association in vivo, we generated transgenic flies expressing a modified NORPA, NORPAC1094S, that lacks the INAD interaction. The transgenic animals display a unique electroretinogram phenotype characterized by slow activation and prolonged deactivation. Double mutant analysis suggests a possible inaccessibility of eye-specific protein kinase C to NORPAC1094S, undermining the observed defective deactivation, and that delayed activation may similarly result from NORPAC1094S being unable to localize in close proximity to the TRP channel. We conclude that INAD acts as a scaffold protein that facilitates NORPA–TRP interactions required for gating of the TRP channel in photoreceptor cells.

Epidermal Growth Factor Signaling and Mitogenesis in Plcg1 Null Mouse Embryonic Fibroblasts

Gene targeting techniques and early mouse embryos have been used to produce immortalized fibroblasts genetically deficient in phospholipase C (PLC)-γ1, a ubiquitous tyrosine kinase substrate. Plcg1−/− embryos die at embryonic day 9; however, cells derived from these embryos proliferate as well as cells from Plcg1+/+ embryos. The null cells do grow to a higher saturation density in serum-containing media, as their capacity to spread out is decreased compared with that of wild-type cells. In terms of epidermal growth factor receptor activation and internalization, or growth factor induction of mitogen-activated protein kinase, c-fos, or DNA synthesis in quiescent cells, PLcg1−/− cells respond equivalently to PLcg1+/+ cells. Also, null cells are able to migrate effectively in a wounded monolayer. Therefore, immortalized fibroblasts do not require PLC-γ1 for many responses to growth factors.

Role of Tyrosine Phosphorylation in Ligand-induced Regulation of Transcytosis of the Polymeric Ig Receptor

The polymeric Ig receptor (pIgR) transcytoses its ligand, dimeric IgA (dIgA), from the basolateral to the apical surface of epithelial cells. Although the pIgR is constitutively transcytosed in the absence of ligand, binding of dIgA stimulates transcytosis of the pIgR. We recently reported that dIgA binding to the pIgR induces translocation of protein kinase C, production of inositol triphosphate, and elevation of intracellular free calcium. We now report that dIgA binding causes rapid, transient tyrosine phosphorylation of several proteins, including phosphatidyl inositol-specific phospholipase C-γl. Protein tyrosine kinase inhibitors or deletion of the last 30 amino acids of pIgR cytoplasmic tail prevents IgA-stimulated protein tyrosine kinase activation, tyrosine phosphorylation of phospholipase C-γl, production of inositol triphosphate, and the stimulation of transcytosis by dIgA. Analysis of pIgR deletion mutants reveals that the same discrete portion of the cytoplasmic domain, residues 727–736 (but not the Tyr734), controls both the ability of pIgR to cause dIgA-induced tyrosine phosphorylation of the phospholipase C-γl and to undergo dIgA-stimulated transcytosis. In addition, dIgA transcytosis can be strongly stimulated by mimicking phospholipase C-γl activation. In combination with our previous results, we conclude that the protein tyrosine kinase(s) and phospholipase C-γl that are activated upon dIgA binding to the pIgR control dIgA-stimulated pIgR transcytosis.

Inhibition of RhoA Translocation and Calcium Sensitization by In Vivo ADP-Ribosylation with the Chimeric Toxin DC3B

Pretreatment of intact rabbit portal vein smooth muscle with the chimeric toxin DC3B (10−6 M, 48 h; Aullo et al., 1993; Boquet et al. 1995) ADP-ribosylated endogenous RhoA, including cytosolic RhoA complexed with rhoGDI, and inhibited the tonic phase of phenylephrine-induced contraction and the Ca2+-sensitization of force by phenylephrine, endothelin and guanosine triphosphate (GTP)γS, but did not inhibit Ca2+-sensitization by phorbol dibutyrate. DC3B also inhibited GTPγS-induced translocation of cytosolic RhoA (Gong et al., 1997a) to the membrane fraction. In DC3B-treated muscles the small fraction of membrane-associated RhoA could be immunoprecipitated, even after exposure to GTPγS, which prevents immunoprecipitation of non-ADP–ribosylated RhoA. Dissociation of cytosolic RhoA–rhoGDI complexes with SDS restored the immunoprecipitability and ADP ribosylatability of RhoA, indicating that both the ADP-ribosylation site (Asn 41) and RhoA insert loop (Wei et al., 1997) are masked by rhoGDI and that the long axes of the two proteins are in parallel in the heterodimer. We conclude that RhoA plays a significant role in G-protein-, but not protein kinase C-mediated, Ca2+ sensitization and that ADP ribosylation inhibits in vivo the Ca2+-sensitizing effect of RhoA by interfering with its binding to a membrane-associated effector.

A novel heat-activated current in nociceptive neurons and its sensitization by bradykinin

Pain differs from other sensations in many respects. Primary pain-sensitive neurons respond to a wide variety of noxious stimuli, in contrast to the relatively specific responses characteristic of other sensory systems, and the response is often observed to sensitize on repeated presentation of a painful stimulus, while adaptation is typically observed in other sensory systems. In most cases the cellular mechanisms of transduction and sensitization in response to painful stimuli are not understood. We report here that application of pulses of noxious heat to a subpopulation of isolated primary sensory neurons rapidly activates an inward current. The ion channel activated by heat discriminates poorly among alkali cations. Calcium ions both carry current and partially suppress the current carried by other ions. The current is markedly increased by bradykinin, a potent algogenic nonapeptide that is known to be released in vivo by tissue damage. Phosphatase inhibitors prolong the sensitization caused by bradykinin, and a similar sensitization is caused by activators of protein kinase C. We conclude that bradykinin sensitizes the response to heat by activating protein kinase C.

Bilirubin, formed by activation of heme oxygenase-2, protects neurons against oxidative stress injury

Heme oxygenase (HO) catalyzes the conversion of heme to carbon monoxide, iron, and biliverdin, which is immediately reduced to bilirubin (BR). Two HO active isozymes exist: HO1, an inducible heat shock protein, and HO2, which is constitutive and highly concentrated in neurons. We demonstrate a neuroprotective role for BR formed from HO2. Neurotoxicity elicited by hydrogen peroxide in hippocampal and cortical neuronal cultures is prevented by the phorbol ester, phorbol 12-myristate 13-acetate (PMA) via stimulation of protein kinase C. We observe phosphorylation of HO2 through the protein kinase C pathway with enhancement of HO2 catalytic activity and accumulation of BR in neuronal cultures. The neuroprotective effects of PMA are prevented by the HO inhibitor tin protoporphyrin IX and in cultures from mice with deletion of HO2 gene. Moreover, BR, an antioxidant, is neuroprotective at nanomolar concentrations.

Dopamine receptor subtypes modulate olfactory bulb γ-aminobutyric acid type A receptors

The γ-aminobutyric acid type A (GABAA) receptor is the predominant Cl− channel protein mediating inhibition in the olfactory bulb and elsewhere in the mammalian brain. The olfactory bulb is rich in neurons containing both GABA and dopamine. Dopamine D1 and D2 receptors are also highly expressed in this brain region with a distinct and complementary distribution pattern. This distribution suggests that dopamine may control the GABAergic inhibitory processing of odor signals, possibly via different signal-transduction mechanisms. We have observed that GABAA receptors in the rat olfactory bulb are differentially modulated by dopamine in a cell-specific manner. Dopamine reduced the currents through GABA-gated Cl- channels in the interneurons, presumably granule cells. This action was mediated via D1 receptors and involved phosphorylation of GABAA receptors by protein kinase A. Enhancement of GABA responses via activation of D2 dopamine receptors and phosphorylation of GABAA receptors by protein kinase C was observed in mitral/tufted cells. Decreasing or increasing the binding affinity for GABA appears to underlie the modulatory effects of dopamine via distinct receptor subtypes. This dual action of dopamine on inhibitory GABAA receptor function in the rat olfactory bulb could be instrumental in odor detection and discrimination, olfactory learning, and ultimately odotopic memory formation.

The neuroprotective effect of pituitary adenylate cyclase-activating polypeptide on cerebellar granule cells is mediated through inhibition of the CED3-related cysteine protease caspase-3/CPP32

Caspase-3 knockout mice exhibit thickening of the internal granule cell layer of the cerebellum. Concurrently, it has been shown that intracerebral injection of pituitary adenylate cyclase-activating polypeptide (PACAP) induces a transient increase of the thickness of the cerebellar cortex. In the present study, we have investigated the possible effect of PACAP on caspase activity in cultured cerebellar granule cells from 8-day-old rat. Incubation of granule neurons with PACAP for 24 h promoted cell survival and prevented DNA fragmentation. Exposure of cerebellar granule cells to the specific caspase-3 inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethylketone (Z-DEVD-FMK) for 24 h markedly enhanced cell survival and inhibited apoptotic cell death. Time-course studies revealed that PACAP causes a prolonged inhibition of caspase-3 activity without affecting caspase-1. Administration of graded concentrations of PACAP for 3 h induced a dose-dependent inhibition of caspase-3 activity. Incubation of granule cells with both dibutyryl-cAMP (dbcAMP) and phorbol 12-myristate 13-acetate (PMA) mimicked the inhibitory effect of PACAP on caspase-3. Cotreatment of cultured neurons with the protein kinase A inhibitor H89 and the protein kinase C inhibitor chelerythrine abrogated the effect of PACAP on caspase-3 activity. In contrast, the ERK kinase inhibitor U0126 did not affect the action of PACAP on caspase-3 activity. These data demonstrate that PACAP prevents cerebellar granule neurons from apoptotic cell death through a protein kinase A- and protein kinase C-dependent inhibition of caspase-3 activity.

Scanning electrochemical microscopy of living cells: Different redox activities of nonmetastatic and metastatic human breast cells

Electrochemical methods have been widely used to monitor physiologically important molecules in biological systems. This report describes the first application of the scanning electrochemical microscope (SECM) to probe the redox activity of individual living cells. The possibilities of measuring the rate and investigating the pathway of transmembrane charge transfer are demonstrated. By this approach, significant differences are detected in the redox responses given by nonmotile, nontransformed human breast epithelial cells, breast cells with a high level of motility (engendered by overexpression of protein kinase Cα), and highly metastatic breast cancer cells. SECM analysis of the three cell lines reveals reproducible differences with respect to the kinetics of charge transfer by several redox mediators.