Neutrophil leukocytes have a pivotal function in innate immunity. Dogma dictates that the lethal blow is delivered to microbes by reactive oxygen species (ROS) and halogens, products of the NADPH oxidase, whose impairment causes immunodeficiency. However, recent evidence indicates that the microbes might be killed by proteases, activated by the oxidase through the generation of a hypertonic, K+-rich and alkaline environment in the phagocytic vacuole. Here we show that K+ crosses the membrane through large-conductance Ca2+-activated K+ (BK(Ca)) channels. Specific inhibitors of these channels, iberiotoxin and paxilline, blocked oxidase-induced 86Rb+ fluxes and alkalinization of the phagocytic vacuole, whereas NS1619, a BK(Ca) channel opener, enhanced both. Characteristic outwardly rectifying K+ currents, reversibly inhibited by iberiotoxin, were demonstrated in neutrophils and eosinophils and the expression of the alpha-subunit of the BK channel was confirmed by western blotting. The channels were opened by the combination of membrane depolarization and elevated Ca2+ concentration, both consequences of oxidase activity. Remarkably, microbial killing and digestion were abolished when the BK(Ca) channel was blocked, revealing an essential and unexpected function for this K+ channel in the microbicidal process.
The effect of anandamide, which activates both the cannabinoid 1 (CB1) receptor and the vanilloid receptor 1 (VR1), was studied on calcitonin gene-related peptide (CGRP) release from cultured primary sensory neurons, the majority of which coexpress the CB1 receptor and VR1. Concentrations of anandamide < 1 micro m produced a small but significant CB1 receptor-mediated inhibition of basal CGRP release while higher concentrations induced VR1-mediated CGRP release. The excitatory effect of anandamide was potentiated by the CB1 receptor antagonist SR141716A. In the presence of SR141716A at concentrations < 100 nm, anandamide was equipotent with capsaicin in stimulating CGRP release. However, at higher concentrations anandamide produced more CGRP release than equimolar concentrations of capsaicin. Three and ten nanomolar anandamide inhibited the capsaicin-evoked CGRP release. In the presence of SR141716A, treatments which activated protein kinase A, protein kinase C and phospholipase C significantly potentiated the anandamide-evoked CGRP release at all anandamide concentrations. Although this potentiation was reduced when the CB1 receptor antagonist was omitted from the buffer, the CGRP release evoked by 300 nm and 1 micro m anandamide was still significantly larger than that seen with nonpotentiated cells. These data indicate that anandamide may regulate CGRP release from capsaicin-sensitive primary sensory neurons in vivo, and that the net effect of anandamide on transmitter release from capsaicin-sensitive primary sensory neurons depends on the concentration of anandamide and the state of the CB1 receptor and VR1. These findings also suggest that anandamide could be one of the molecules responsible for the development of inflammatory heat hyperalgesia.
Calcium plays a regulatory role in several aspects of protein trafficking in the cell. Both vesicle fusion and vesicle formation can be inhibited by the addition of calcium chelators. Because the effects of calcium chelators have been studied predominantly in cell-free systems, it is not clear exactly which transport steps in the secretory pathway are sensitive to calcium levels. In this regard, we have studied the effects of calcium chelators on both anterograde and retrograde protein transport in whole cells. Using both cytochemical and biochemical analyses, we find that the anterograde-directed exit of vesicular stomatitis virus G protein and the retrogradedirected exit of Shiga toxin from the Golgi apparatus are both inhibited by calcium chelation. The exit of vesicular stomatitis virus G from a pre-Golgi compartment and the exit of Shiga toxin from an endosomal compartment are sensitive to the membrane-permeant calcium chelator 1,2-bis(2-amino phenoxy)ethane-N,N,N,N-tetraacetic acid-tetrakis (acetoxymethyl ester) (BAPTA-AM). By contrast, endoplasmic reticulum exit and endocytic internalization from the plasma membrane are not affected by BAPTA. Together, our data show that some, but not all, trafficking steps in the cell may be regulated by calcium. These studies provide a framework for a more detailed analysis of the role of calcium as a regulatory agent during protein transport.
The inhibitory cannabinoid 1 receptor and the excitatory vanilloid receptor 1, both of which are responsive to the endogenous ligand anandamide, are co-expressed on a subpopulation of primary sensory neurones. We report that activation of the cannabinoid 1 receptor/vanilloid receptor 1-co-expressing primary sensory neurones induces the production and release of anandamide. Application of capsaicin (3 nM)1 lM) to cultured primary sensory neurones evoked calcitonin gene-related peptide release, which was significantly increased by the selective cannabinoid 1 receptor antagonist, SR141716A (200 nM). Mass spectrometric analyses of the extracellular solution revealed that exposure to capsaicin (10 nM or 100 nM) enhanced the anandamide concentration of the medium from less then 0.05 pmol/lL to more then 2 pmol/lL. Depolarization of the neurones with 50 mM KCl also enhanced the anandamide content of the buffer. Both the capsaicin-and KCl-induced anandamide release depended on extracellular Ca 2+ . Prolonged treatment of the cultures with capsaicin (10 lM) reduced both the capsaicin-and KClinduced anandamide release. These findings indicate that activation of capsaicin-sensitive primary sensory neurones evokes anandamide production and release, and that anandamide might be a key endogenous regulator of the excitability of these neurones. Anandamide activates two receptors, the cannabinoid 1 (CB 1 ) receptor and the vanilloid receptor 1 (VR1) (Devane et al. 1992;Zygmunt et al. 1999). The CB 1 receptor is expressed in a number of areas in the nervous system including dorsal root ganglion neurones (Ahluwalia et al. 2000), and its activation produces various effects including anti-nociception (Calignano et al. 1998). At the cellular level activation of the CB 1 receptor reduces adenylate cyclase activity and Ca 2+ currents, and increases K + currents, which result in reduced membrane excitability and transmitter release (Mackie et al. 1995;Twitchell et al. 1997;Kathmann et al. 1999). In contrast, VR1 is almost exclusively expressed by the two subpopulations of nociceptive primary sensory neurones, the so-called peptidergic and Bandeiraea simplicifolia isolectin B4-binding cells (Caterina et al. 1997;Guo et al. 1999;Michael and Priestley 1999). Activation of VR1, for example by capsaicin, excites and evokes the release of peptides, such as calcitonin gene-related peptide (CGRP), from the capsaicin-sensitive cells (Caterina et al. 1997;Tognetto et al. 2001).We have shown recently that all VR1-expressing primary sensory neurones also express the CB 1 receptor (Ahluwalia et al. 2000). In agreement with this co-expression, activation of the CB 1 receptor reduces the capsaicin-induced transmitter release from primary sensory neurones (Richardson et al.
Abstract. The intrahepatic distribution of apolipoprotein E has been assessed by immunogold labeling of cryosections as well as by Western blotting of organelles isolated from liver homogenates. Both techniques supported the prior analytical fractionation studies of Wong (1989) who concluded that intrahepatic apoE was largely endosomal. All endosomal components decorated by gold particles indicative of apoE antigenicity in cryosections appeared filled with lipoprotein-like particles thereby accounting for this prominent morphological feature of isolated liver endosomes. The distribution of gold particles about the hepatic Golgi apparatus revealed a high content of apoE in closely apposed endosomes, ca. 400 nm in diameter, double labeled for apoE and internalized HRP. Remarkably, apoE (but not internalized HRP) was also observed within saccular distensions of all saccules of stacked Golgi cisternae but absent from the flattened saccular components as was also observed for apoB. This contrasted with albumin, the major secretory protein, which was uniformly distributed throughout the hepatic Golgi apparatus. These observations support a growing body of evidence for intra-Golgi sorting of secretory material in hepatic Golgi apparatus. The lack of any immunoreactive apoE or albumin in small 70-90 nm vesicles about the Golgi cisternae suggests limits to current models of vesicle-mediated intra-Golgi transport.
Calcium has been implicated in regulating vesicle fusion reactions, but its potential role in regulating other aspects of protein transport, such as vesicle assembly, is largely unexplored. We find that treating cells with the membrane-permeable calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), leads to a dramatic redistribution of the vesicle coat protein, coatomer, in the cell. We have used the cell-free reconstitution of coatprotomer I (COPI) vesicle assembly to characterize the mechanisms of this redistribution. We find that the recovery of COPI-coated Golgi vesicles is inhibited by the addition of BAPTA to the cell-free vesicle budding assay. When coatomer-coated membranes are incubated in the presence of calcium chelators, the membranes "uncoat," indicating that calcium is necessary for maintaining the integrity of the coat. This uncoating is reversed by the addition of calcium. Interestingly, BAPTA, a calcium chelator with fast binding kinetics, is more potent at uncoating the coatomer-coated membrane than EGTA, suggesting that a calcium transient or a calcium gradient is important for stabilizing COPI vesicle coat. The primary target for the effects of calcium on coatomer recruitment is a step that occurs after ADP-ribosylation factor binding to the membrane. We suggest that a calcium gradient may serve to regulate the timing of vesicle uncoating.
The synthesis and secretion of enamel proteins (EPs) in rat incisors was examined using cytochemical and biochemical methods. Radioautography after injection of 3H-methionine showed that ameloblasts in the presecretory, secretory, and maturation stages of amelogenesis actively synthesized and secreted proteins. Immunocytochemistry with an antibody to mouse amelogenins revealed the presence of EPs in the protein synthetic and secretory organelles of these cells at all three stages. Labeling was also found in elements of the endosomal/lysosomal compartment. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining of proteins extracted from enamel and enamel organ showed several protein bands. However, transfer to nitrocellulose paper and immunoblotting revealed that most of the proteins recognized by the antibody were situated between approximately 14 and 32 kDa. EPs were further characterized by using lectins to examine their carbohydrate content. Lectin-gold cytochemistry on sections showed the binding of wheat germ agglutinin and Helix pomatia lectin to secretory stage enamel. Lectin blotting indicated that the amelogenins were heterogeneously glycosylated and contained the sugars N-acetyl-glucosamine/N-acetyl-neuraminic acid and N-acetyl-D-galactosamine. Fluorography at 6 and 10 min and 1 h after injection of 35S-methionine revealed four labeled bands in the main amelogenin group near 22, 28, 30, and 32 kDa. A short-lived protein of approximately 58 kDa was also observed primarily in cells. The appearance of labeled proteins in enamel was paralleled by their disappearance from cells and the intensity of the radiolabeled protein bands, both, in enamel and in cells, decreased towards the maturation stage. These data are consistent with the concept that ameloblasts produce multiple amelogenins throughout amelogenesis.
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