Presence Of Extracellular Divalent Cations Research Articles

Permeation Properties of CALHM1

Department of Physiology, University of Pennsylvania, Philadelphia, PA 19104Recently, CALHM1, a gene of previously unknown function, was identified as a risk factor for late-onset Alzheimer‘s disease (AD). It was suggested that CALHM1 might be an ion channel or ion channel regulator. We used two-electrode voltage clamp (TEV) to measure CALHM1-induced ionic conductances in the plasma membrane of Xenopus oocytes. To investigate which ions permeate the CALHM1-induced conductance, we employed instantaneous current/voltage protocols to measure reversal potentials (Erev) in various solutions. In the absence of extracellular divalent cations, CALHM1 is weakly cation selective, with PNa : PK : PCl = 1 : 1.11 : 0.52. In CALHM1 injected oocytes, the relative permeability among monovalent cations (with respect to Na+) is PRb = PCs = PK > PNa > PLi > PNMDG, indicating that CALHM1-induced permeability is relatively nonselective for monovalent cations. CALHM1 induced currents are divalent cation selective, with PNa : PCa : PBa : PMg = 1 : 13.8 : 8.6 : 3.1. This selectivity sequence represents a Sherry III/IV sequence, consistent with a weak-field strength site in the permeation pore. The presence of extracellular divalent cations strongly alters the gating properties of CALHM1 induced currents. However, the presence of extracellular divalent cations only slightly alters the relative permeability of K+ and Cl- (PNa : PK : PCl = 1 : 1.46 : 0.88). Using large organic cations and a volume exclusion model, we estimated the radius of the CALHM1 associated ion channel pore in the absence of divalent cations to be 6.9 to 8.7 A. (Supported by NIH GM/DK56328 and a pilot project from the University Pennsylvania AD Core Center.)

Comparison of the Pseudomonas aeruginosa andEscherichia coli PhoQ Sensor Domains: EVIDENCE FOR DISTINCT MECHANISMS OF SIGNAL DETECTION

The PhoP-PhoQ two-component system is present in a number of Gram-negative bacteria where it has roles in Mg(2+) homeostasis and virulence. PhoQ is a transmembrane histidine kinase that activates PhoP-mediated regulation of a set of genes when the extracellular concentration of divalent cations is low. Divalent cations are thought to interact directly with the periplasmic PhoQ sensor domain. The PhoP-PhoQ systems of Escherichia coli and Pseudomonas aeruginosa are similar in their biological response to extracellular divalent cations; however, their sensor domains display little sequence identity. Here we have begun to explore the consequences of this sequence divergence by comparing the biophysical properties of the P. aeruginosa PhoQ sensor domain with the corresponding E. coli sensor domain. Unlike the E. coli protein, the P. aeruginosa PhoQ sensor domain undergoes changes in the circular dichroism and fluorescence spectra as well as destabilization of its dimeric form in response to divalent cations. These results suggest that distinct mechanisms of signal detection are utilized by these proteins. A hybrid protein in which the E. coli sensor domain has been substituted with the corresponding P. aeruginosa sensor domain responds normally to the presence of extracellular divalent cations in vivo in E. coli. Thus, despite apparent differences in the structural response to its stimulus, the P. aeruginosa sensor domain transduces signals to the E. coli PhoQ cytoplasmic kinase domain in a manner that mimics normal E. coli PhoQ function.

Genomic structure, developmental distribution and functional properties of the chicken P2X(5) receptor.

We report here the cloning of a chicken cDNA (402 aa) showing high sequence similarity to the previously cloned rat and human P2X(5) receptors (67 and 69%, respectively). The chicken P2X(5) subunit is encoded by a gene composed of 12 translated exons, which shows conserved genomic structure with mammalian P2X genes. In HEK-293 cells heterologously expressing chicken P2X(5) receptors, ATP activates a current that desensitizes in a way that is dependent on the presence of extracellular divalent cations. ATP and 2-methylthio ATP are equipotent agonists (EC(50) approximately 2 microM) and suramin and pyridoxal 5-phosphate-6-azophenyl-2',4'-disulfonic acid are potent antagonists. Additionally, reversal potential measurements indicate that chicken P2X(5) is permeable not only to cations but also to chloride (P(Cs+)/P(Cl-) approximately 1.9), as has been described for native P2X receptor mediated responses in embryonic chicken skeletal muscle. mRNA distribution of chicken P2X(5) was determined by in situ hybridization analysis in both whole embryos and on tissue slices of heart and skeletal muscle. Our results suggest that chicken P2X(5) receptors are expressed in developing muscle and might play a role in early muscle differentiation.

Regulation of activation of bovine neutrophils by aggregated immunoglobulin G

Abstract Objectives To investigate the role of extracellular Ca2+ and Mg2+ in aggregated IgG (algG)-mediated cellular activation, and to determine how algG-induced activation is coupled to Ca2+ homeostasis in bovine neutrophils. Sample Population 4 clinically normal, lactating Holstein cows, in their second lactation, which ranged between 60 and 150 days. Procedure algG was prepared by heating bovine igG, and C5a was obtained by activating fetal bovine serum with zymosan. Luminol-amplified chemiluminescence (CL) of isolated neutrophils was measured in the presence of algG or phorbol 12-myristate 13-acetate (PMA). The reaction mixture contained either Hanks’ balanced salt solution or Ca2+- and Mg2+-free Hanks’ balanced salt solution. Binding of algG to neutrophils was measured by flow cytometry after incubation with fluorescein isothiocyanate-conjugated second antibody. Intracellular-free concentration [Ca2+] was measured in a fluorescence spectrofluorometer after incubation of neutrophils, loaded with the fluorescent dye fura-2 acetoxymethyl ester, with either algG or C5a. Results In a Ca2+- and Mg2+-containing reaction mixture, algG induced strong CL responses, whereas removal of extracellular divalent cations almost abolished the respiratory burst activity. The CL emission on stimulation with PMA was independent of extracellular Ca2+ and Mg2+. Examination of cells by flow cytometry after incubation with algG indicated that the binding of algG was identical in the presence and absence of extracellular Ca2+ and Mg2+. No increase in [Ca2+] was seen in fura-2 acetoxymethylester-loaded neutrophils after stimulation with algG. C5a induced a typical transient increase in [Ca2+]. Conclusions algG-induced activation of bovine neutrophils is highly dependent on presence of extracellular divalent cations. This dependency is not caused by the need of divalent cations for binding of algG by neutrophils or because the influx of Ca2+ from the extracellular space is an integral component of algG-mediated activation pathway. Because need for extracellular Ca2+ and Mg2+ could be partially circumvented by pretreating neutrophils with PMA, it is possible that this activation pathway may involve a protein kinase C, which is not directly coupled to receptors for algG. (Am J Vet Res 1996;57:1312-1316)

Activated Conformations of Very Late Activation Integrins Detected by a Group of Antibodies (HUTS) Specific for a Novel Regulatory Region(355-425) of the Common β1 Chain

The very late activation antigens (VLA) or beta 1 integrins mediate cell attachment to different extracellular matrix proteins and intercellular adhesions. The ligand binding activity of these adhesion receptors is not constitutive and can be regulated by temperature, presence of extracellular divalent cations, stimulatory monoclonal antibodies (mAbs), and cellular activation. We have generated three novel mAbs, HUTS-4, HUTS-7, and HUTS-21, recognizing specific epitopes on the common beta 1 subunit (CD29) of VLA integrins whose expression correlates with the ligand binding activity of these heterodimeric glycoproteins. This correlation has been demonstrated for several integrin heterodimers in different cell systems using a variety of extracellular and intracellular stimuli for integrin activation. Thus, the presence of micromolar concentrations of extracellular Mn2+, preincubation with the activating anti-beta 1 mAb TS2/16, and cell treatment with phorbol esters or calcium ionophores, induced the expression of the HUTS beta 1 epitopes on T lymphoblasts. Using a panel of human-mouse beta 1 chimeric molecules, we have mapped these epitopes to the 355-425 sequence of the beta 1 polypeptide. This segment represents therefore a novel regulatory region of beta 1 that is exposed upon integrin activation. Interestingly, binding of HUTS mAbs to partially activated VLA integrins results in maximal activation of these adhesion receptors and enhancement of cell adhesion to beta 1 integrin ligands collagen, laminin, and fibronectin.

Osteopontin-Stimulated Vascular Smooth Muscle Cell Migration Is Mediated by β 3 Integrin

Osteopontin (OPN), a 41-kDa phosphorylated glycoprotein, has been detected in rat aorta and carotid arteries, and expression of its mRNA in blood vessels is strongly increased in response to vascular injury. To investigate the potential role of OPN in vascular pathophysiology, we studied the effect of rat OPN on aortic smooth muscle cell migration and proliferation in vitro. OPN enhanced the migration of rat smooth muscle cells in a time- and concentration-dependent manner with an EC 50 value of 46 ± 11 nmol/liter ( n = 5). The maximal increase in cell migration by OPN was 29-fold over basal levels. OPN-induced smooth muscle cell migration was inhibited in a concentration-dependent manner by the monoclonal antibody F11, which recognizes the rat integrin subunit β 3. In contrast, polyclonal antiserum recognizing the rat integrin β 1 subunit did not inhibit smooth muscle cell migration in response to OPN, but did block fibronectin-promoted migration. Moreover, OPN-induced smooth muscle cell migration was dependent on the presence of extracellular divalent cations and was significantly inhibited by anti-OPN antibodies. OPN did not stimulate [ 3H]thymidine incorporation into cultured smooth muscle cells, indicating that it selectively enhanced migration. In view of the pathological significance of arterial smooth muscle cell migration in the formation of intimal thickening, our results suggest that smooth muscle cell recognition of OPN, probably through the vitronectin receptor, α vβ 3, could play a role in the cells' response to vascular injury and especially neointima formation.

Evidence for a role of glycoprotein IIb-IIIa, distinct from its ability to support aggregation, in platelet activation by ionophores in the presence of extracellular divalent cations

Ionophore A23187-induced 14C-serotonin secretion and thromboxane B2(TxB2) formation were found to be absent in citrated platelet-rich plasma (PRP) from thrombasthenic subjects and in normal PRP treated with glycoprotein (GP) IIb-IIIa complex-specific monoclonal antibodies. Both responses were restored to normal levels when 5 mmol/L EDTA was present, indicating that their absence was not caused by the absence of aggregation per se. In gel-filtered platelets (GFP) incubated with various additions, the blockade or absence of GPIIb-IIIa resulted in reduced A23187-induced secretion and TxB2 formation in media containing 1 mmol/L Ca2+ with or without fibrinogen and 1 mmol/L Mg2+ plus fibrinogen, but not when Ca, Mg-free buffer alone, 1 mmol/L EDTA, or fibrinogen alone were present. In contrast, no such dependence or GPIIb- IIIa was seen in GFP stimulated with thrombin or phorbol myristate acetate in the presence of 1 mmol/L Ca2+, 1 mmol/L EDTA, or buffer alone. The inhibition of ionophore-induced responses seen in both normal GFP treated with antibodies and thrombasthenic GFP was not associated with any significant alteration of the ionophore-mediated [Ca2+]i increase, as measured in both aequorin-loaded GFP stimulated with A23187 and fura-2-loaded GFP stimulated with ionomycin. Incubation of normal GFP with either the monoclonal antibodies or the ligand binding site peptide RGDS in the presence of 1 mmol/L Ca2+ caused virtually complete inhibition of A23187-induced aggregation, measured as the loss of single platelets, but RGDS, in contrast to the antibodies, did not inhibit secretion or TxB2 formation. We conclude that platelet activation induced by ionophores in the presence, but not in the absence, of extracellular divalent cations involves a GPIIb-IIIa- dependent process that most likely involves a property of the ligand- occupied form of the complex distinct from its ability to support aggregation. This could represent another example of an aggregation- independent activity of the receptor-occupied state of the GPIIb-IIIa complex in signal transduction.

Evidence for a Role of Glycoprotein IIb-IIIa, Distinct From Its Ability to Support Aggregation, in Platelet Activation by Ionophores in the Presence of Extracellular Divalent Cations

AbstractIonophore A23187-induced 14C-serotonin secretion and thromboxane B2(TxB2) formation were found to be absent in citrated platelet-rich plasma (PRP) from thrombasthenia subjects and in normal PRP treated with glycoprotein (GP) IIb-IIIa complex-specific monoclonal antibodies. Both responses were restored to normal levels when 5 mmol/L EDTA was present, indicating that their absence was not caused by the absence of aggregation per se. In gel-filtered platelets (GFP) incubated with various additions, the blockade or absence of GPIIb-IIIa resulted in reduced A23187-in-duced secretion and TxB2 formation in media containing 1 mmol/L Ca2+ with or without fibrinogen and 1 mmol/L Mg2+ plus fibrinogen, but not when Ca, Mg-free buffer alone, 1 mmol/L EDTA, or fibrinogen alone were present. In contrast, no such dependence or GPIIb-llla was seen in GFP stimulated with thrombin or phorbol myristate acetate in the presence of 1 mmol/L Ca2+, 1 mmol/L EDTA, or buffer alone. The inhibition of ionophore-induced responses seen in both normal GFP treated with antibodies and thrombasthenic GFP was not associated with any significant alteration of the ionophore-mediated [Ca2+], increase, as measured in both aequorin-loaded GFP stimulated with A23187 and fura-2-loaded GFP stimulated with ionomycin. Incubation of normal GFP with either the monoclonal antibodies or the ligand binding site peptide RGDS in the presence of 1 mmol/L Ca2+ caused virtually complete inhibition of A23187-induced aggregation, measured as the loss of single platelets, but RGDS, in contrast to the antibodies, did not inhibit secretion or TxB2 formation. We conclude that platelet activation induced by ionophores in the presence, but not in the absence, of extracellular divalent cations involves a GPIIb-llla-depen-dent process that most likely involves a property of the lig-and-occupied form of the complex distinct from its ability to support aggregation. This could represent another example of an aggregation-independent activity of the receptor-occupied state of the GPIIb-IIIa complex in signal transduction.

Modulation of Neutrophil Oxidative Responses to Soluble Stimuli by Platelet-Activating Factor

Abstract The role of platelet activating factor (PAF) as a regulator of human neutrophil superoxide (O2-) generation in response to soluble and particulate stimuli was examined. At concentrations greater than 10(-7) mol/L, PAF alone induced a brief burst of O2- production. When cells were exposed to PAF and either the chemotactic peptide n-formyl- methionyl-leucyl-phenylalanine (FMLP 10(-7) mol/L) or the tumor promoter phorbol myristate acetate (PMA 10 ng/mL), a marked synergistic augmentation of O2- release was noted when compared to control cells stimulated with FMLP or PMA alone. Mean percentage of enhancement by 10(-5) mol/L of PAF was 297% +/- 35% (n = 9) of control responses to FMLP and 185% +/- 16% (n = 3) of control responses to PMA. Consistent enhancement occurred with PAF concentrations of as low as 10(-9) mol/L. Enhancement could be demonstrated when neutrophils were exposed to PAF either at the same time as, or up to 60 minutes prior to, the second stimulus, and was neither reversed by removal of PAF from the medium prior to addition of FMLP or PMA nor dependent on the presence of extracellular divalent cations. Continuous recordings revealed that the enhancement was due to an increased maximal rate of O2- production. In contrast, PAF concentrations up to 10(-5) mol/L had only a minimal effect on the response to neutrophils to opsonized zymosan. Analysis of the enhancing properties of lipids structurally related to PAF revealed that the critical moiety was the saturated fatty acid at position 1. These results indicate the presence of a PAF-mediated positive feedback loop whereby the oxidative burst induced by some soluble stimuli is augmented. Modulation of neutrophil O2- production by PAF may serve to amplify neutrophil oxidative responses at sites of inflammation.

Modulation of neutrophil oxidative responses to soluble stimuli by platelet-activating factor

The role of platelet activating factor (PAF) as a regulator of human neutrophil superoxide (O2-) generation in response to soluble and particulate stimuli was examined. At concentrations greater than 10(-7) mol/L, PAF alone induced a brief burst of O2- production. When cells were exposed to PAF and either the chemotactic peptide n-formyl-methionyl-leucyl-phenylalanine (FMLP 10(-7) mol/L) or the tumor promoter phorbol myristate acetate (PMA 10 ng/mL), a marked synergistic augmentation of O2- release was noted when compared to control cells stimulated with FMLP or PMA alone. Mean percentage of enhancement by 10(-5) mol/L of PAF was 297% +/- 35% (n = 9) of control responses to FMLP and 185% +/- 16% (n = 3) of control responses to PMA. Consistent enhancement occurred with PAF concentrations of as low as 10(-9) mol/L. Enhancement could be demonstrated when neutrophils were exposed to PAF either at the same time as, or up to 60 minutes prior to, the second stimulus, and was neither reversed by removal of PAF from the medium prior to addition of FMLP or PMA nor dependent on the presence of extracellular divalent cations. Continuous recordings revealed that the enhancement was due to an increased maximal rate of O2- production. In contrast, PAF concentrations up to 10(-5) mol/L had only a minimal effect on the response to neutrophils to opsonized zymosan. Analysis of the enhancing properties of lipids structurally related to PAF revealed that the critical moiety was the saturated fatty acid at position 1. These results indicate the presence of a PAF-mediated positive feedback loop whereby the oxidative burst induced by some soluble stimuli is augmented. Modulation of neutrophil O2- production by PAF may serve to amplify neutrophil oxidative responses at sites of inflammation.

Phorbol 12,13-dibutyrate (P(Bu) 2)-treated human blood mononuclear cells bind to each other

Treatment of human blood mononuclear cells with nanomolar concentrations of phorbol 12,13-dibutyrate (P(Bu) 2) induced their aggregation. The phenomenon was seen within a few minutes and reached its maximum manifestation after 20 min. At this time, 20–30% of unfractionated and nylon wool-passed mononuclear leukocytes were in the aggregates. The influence of pretreatment with 2-deoxyglucose, NaN 3, EDTA, cyclohexamide, or incubation at 4 °C on the phenomenon indicated that it is energy and temperature dependent, requires the presence of extracellular divalent cations, and is independent of protein synthesis. Nonaggregating 2-deoxyglucose- and NaN 3-pretreated cells could still bind [ 3H]P(Bu) 2 which rules out the possibility that the phorbol ester molecule acts as a bridge between the aggregated cells. Seventeen percent of the P(Bu) 2-treated T-cell population bound untreated autologous and allogeneic cells. The binding property has a certain species specificity because only 4% of the cells interacted with mouse lymphocytes. At the ultrastructural level, the intercellular binding showed broad areas of surface contact (both between lymphocytes and lymphocyte-monocyte) and “trapping” by surface processes was not seen. Aggregated cells did not show cytopathogenic changes.

PGBX, a prostagandin derivative, mimics the action of the calcium ionophore A23187 on human neutrophils.

The interactions have been studied of a water-soluble, polymeric derivative of prostaglandin B1, PGBX, with human polymorphonuclear leukocytes (PMN). PGBX, which is a potent ionophore of divalent cations, provoked superoxide anion (O2.-) generation and lysosomal enzyme release in cytochalasin B-treated PMN in the presence of extracellular divalent cations (Ca2+, Sr2+, Mg2+, Mn2+, Ba2+). Kinetic and dose-response studies showed that PGBX mimicked te action of ionophore A23187 in PMN. Both ionophores induced superoxide generation and release of enzymes from specific and azurophil granules (lysozyme > beta-glucuronidase) without provoking release of the cytoplasmic marker enzyme lactic dehydrogenase. In contrast, the precursor of PGBX, prostaglandin B1 (PGB1), and arachidonate did not mimic ionophore-induced stimulation of PMN. PGBX induced enzyme release both in the presence of extracellular Ca2+ and Ba2+ (both of which it translocates in model liposomes), whereas A23187 showed specificity for Ca2+ (which it translocates preferentially over Ba2+). These studies indicate that the actions of a water-soluble polymer (PGBX) derived from a naturally occurring prostaglandin (PGB1) on human neutrophils resemble those of a classical ionophore (A23187). Moreover, they provide additional evidence that increments in the intracellular levels of divalent cations may signal stimulus-secretion coupling in human neutrophils.