Biomedical subjects

S V Pizzo

Publications and source records attributed to S V Pizzo.

At least 37 records · Page 2Linked to original sources

PubMed1999

Mechanism of hypochlorite-mediated inactivation of proteinase inhibition by alpha 2-macroglobulin.

The proteinase-proteinase inhibitor balance plays an important role in mediating inflammation-associated tissue destruction. alpha 2-Macroglobulin (alpha 2M) is a high-affinity, broad-spectrum proteinase inhibitor found abundantly in plasma and interstitial fluids. Increased levels of alpha 2M and proteinase-alpha 2M complexes can be demonstrated in patients with sepsis, emphysema, peridontitis, rheumatoid arthritis, and other inflammatory diseases. Despite these increased levels, proteolysis remains a significant problem. We hypothesized that a mechanism for inactivating alpha 2M-mediated proteinase inhibition must exist and recently demonstrated that alpha 2M isolated from human rheumatoid arthritis synovial fluid is oxidized and has decreased functional activity. The oxidant responsible for alpha 2M inactivation and the mechanism of such destruction were not studied. We now report that while hypochlorite and hydroxyl radical both modify amino acid residues on alpha 2M, only hypochlorite can abolish the ability of alpha 2M to inhibit proteinases. Hydrogen peroxide, on the other hand, has no effect on alpha 2M structure or function. Protein unfolding with increased susceptibility to proteolytic cleavage appears to be involved in alpha 2M inactivation by oxidation. The in vivo relevance of this mechanism is supported by the presence of multiple cleavage fragments of alpha 2M in synovial fluid from patients with rheumatoid arthritis, where significant tissue destruction occurs, but not in patients with osteoarthritis. These results provide strong evidence that hypochlorite oxidation contributes to enhanced tissue destruction during inflammation by inactivating alpha 2M.

Arthritis, Rheumatoid↗

PubMed1999

Coordinate regulation of the alpha(2)-macroglobulin signaling receptor and the low density lipoprotein receptor-related protein/alpha(2)-macroglobulin receptor by insulin.

We have studied insulin-dependent regulation of macrophage alpha(2)-macroglobulin signaling receptors (alpha(2)MSR) and low density lipoprotein receptor-related protein/alpha(2)M receptors (LRP/alpha(2)MR) employing cell binding of (125)I-alpha(2)M*, inhibition of binding by receptor-associated protein (RAP) or Ni(2+), LRP/alpha(2)MR mRNA levels, and generation of second messengers. Insulin treatment increased the number of alpha(2)M* high (alpha(2)MSR) and low (LRP/alpha(2)MR) affinity binding sites from 1, 600 and 67,000 to 2,900 and 115,200 sites per cell, respectively. Neither RAP nor Ni(2+) blocked the binding of (125)I-alpha(2)M* to alpha(2)MSR on insulin- or buffer-treated cells, but they both blocked binding to LRP/alpha(2)MR. Insulin significantly increased LRP/alpha(2)MR mRNA levels in a dose- and time-dependent manner. Insulin-augmented (125)I-alpha(2)M* binding to macrophages was severely reduced by wortmannin, LY294002, PD98059, SB203580, or rapamycin. The increase in alpha(2)MSR receptor synthesis was reflected by augmented generation of IP(3) and increased [Ca(2+)](i) levels upon receptor ligation. Incubation of macrophages with wortmannin, LY294002, PD98059, SB203580, rapamycin, or antibodies against insulin receptors before insulin treatment and alpha(2)M* stimulation significantly reduced the insulin-augmented increase in IP(3) and [Ca(2+)](i) levels. Pretreatment of cells with actinomycin D or cycloheximide blocked the synthesis of new alpha(2)MSR. In conclusion, we show here that insulin coordinately regulates macrophage alpha(2)MSR and LRP/alpha(2)MR, utilizing both the PI 3-kinase and Ras signaling pathways to induce new synthesis of these receptors.

Animals↗

PubMed1999

Incorporation of non-proteolytic proteins by murine alpha2-macroglobulin.

Human alpha2-macroglobulin is a tetrameric glycoprotein with a molecular weight of 718 kDa that is present in human plasma at high concentrations. Murine alpha2-macroglobulin is homologous to human alpha2-macroglobulin but it undergoes post-translational cleavage in the subunits. Each subunit of alpha2-macroglobulin contains a thiolester which can be cleaved by small nucleophiles. In human alpha2-macroglobulin this results in a conformational change to a receptor-recognized form and a change in the electrophoretic mobility. Recent work has demonstrated that this process is reversible and during this reversal non-proteolytic proteins can become covalently trapped within the human alpha2-macroglobulin molecule. The present study further investigates this observation and examines the question whether reversal of thiolester cleavage occurs in mouse alpha2-macroglobulin. Previous studies suggest that small nucleophiles only partially convert mouse alpha2-macroglobulin to a receptor-recognized form. We demonstrate here that under appropriate conditions, mouse alpha2-macroglobulin is fully converted by NH3. We also demonstrate that despite structural and kinetic differences between human and mouse alpha2-macroglobulin, both molecules are able to incorporate non-proteolytic ligands in a similar manner. This leads us to propose a general model of ligand incorporation via nucleophilic exchange in multimeric alpha-macroglobulins.

Ammonia↗

PubMed1999

Angiostatin binds ATP synthase on the surface of human endothelial cells.

Angiostatin, a proteolytic fragment of plasminogen, is a potent antagonist of angiogenesis and an inhibitor of endothelial cell migration and proliferation. To determine whether the mechanism by which angiostatin inhibits endothelial cell migration and/or proliferation involves binding to cell surface plasminogen receptors, we isolated the binding proteins for plasminogen and angiostatin from human umbilical vein endothelial cells. Binding studies demonstrated that plasminogen and angiostatin bound in a concentration-dependent, saturable manner. Plasminogen binding was unaffected by a 100-fold molar excess of angiostatin, indicating the presence of a distinct angiostatin binding site. This finding was confirmed by ligand blot analysis of isolated human umbilical vein endothelial cell plasma membrane fractions, which demonstrated that plasminogen bound to a 44-kDa protein, whereas angiostatin bound to a 55-kDa species. Amino-terminal sequencing coupled with peptide mass fingerprinting and immunologic analyses identified the plasminogen binding protein as annexin II and the angiostatin binding protein as the alpha/beta-subunits of ATP synthase. The presence of this protein on the cell surface was confirmed by flow cytometry and immunofluorescence analysis. Angiostatin also bound to the recombinant alpha-subunit of human ATP synthase, and this binding was not inhibited by a 2,500-fold molar excess of plasminogen. Angiostatin's antiproliferative effect on endothelial cells was inhibited by as much as 90% in the presence of anti-alpha-subunit ATP synthase antibody. Binding of angiostatin to the alpha/beta-subunits of ATP synthase on the cell surface may mediate its antiangiogenic effects and the down-regulation of endothelial cell proliferation and migration.

Adenosine Triphosphatases↗

PubMed1999

Upregulation of macrophage plasma membrane and nuclear phospholipase D activity on ligation of the alpha2-macroglobulin signaling receptor: involvement of heterotrimeric and monomeric G proteins.

The effect of ligating the alpha2-macroglobulin signaling receptor (alpha2MSR) with receptor-recognized forms of alpha2M (alpha2M*) was studied with respect to phospholipase D (PLD) activity in murine macrophages, their plasma membranes, and nuclei. PLD activity in plasma membranes and nuclei increased linearly up to a ligand concentration of about 100 pM of either alpha2M* or a cloned and expressed receptor binding fragment (RBF). The RBF binding site mutant K1370A, which binds with high affinity to alpha2MSR, also increased nuclear PLD activity comparable to RBF and alpha2M*. Phorbol dibutyrate caused a two- to threefold stimulation of membrane and nuclear PLD activity, whereas PLD activity was nearly abolished by downregulation of protein kinase C; prior treatment with staurosporin, genestein, cyclosporin A, actinomycin D; or chelation of intracellular Ca2+. In permeabilized macrophages, isolated plasma membranes, and nuclei, GTP-gamma-S increased alpha2M*-stimulated PLD activity via a pertussis toxin-insensitive G protein and this effect was abolished on preincubation with GDP-beta-S. Incubation of plasma membranes with polyclonal antibody against sARFII, or the addition of cytosol which was immunoprecipitated with antibody against sARFII, greatly reduced alpha2M*-stimulated PLD activity in the presence of GTP-gamma-S. Preincubation of plasma membranes with GDP-beta-S prior to the addition of GTP-gamma-S and recombinant ARF1 significantly inhibited alpha2M*-stimulation of PLD activity. Nuclear PLD activity was maximally stimulated in the presence of both GTP-gamma-S and rARF1, whereas plasma membrane PLD activity was maximally stimulated in the presence of rARF1, GTP-gamma-S, RhoA, and ATP. In contrast, nuclear PLD activity was not affected by RhoA either alone or in combination with GTP-gamma-S or ATP.

ADP-Ribosylation Factor 1↗

PubMed1999

Exposure of cultured murine peritoneal macrophages to low concentrations of beryllium induces increases in intracellular calcium concentrations and stimulates DNA synthesis.

Exposure of humans to beryllium dusts can induce a specific form of chronic pneumonitis that consists mainly of noncaseating granulomas in the lungs. Multiple studies have documented both genetic and immune components of chronic berylliosis. Much work has focused on T cells and their reactivity in berylliosis, but less work has focused on the end effector cells in granulomatous inflammation, macrophages. Because macrophages must become activated to form granulomas, and they become activated by responding to numerous immunomodulatory signals, we investigated the effects of beryllium (BeCl2) on a central signal transduction pathway in macrophages, increases in intracellular calcium ([Ca2+]i). Exposure of cultured murine peritoneal macrophages to low, nontoxic concentrations induced successive spikes or oscillations in [Ca2+]i. Concentrations as low as 5 nM induced significant increases in [Ca2+]i. The source of the increased [Ca2+]i was exclusively extracellular in that increases in [Ca2+]i could be completely blocked by chelating extracellular Ca2+, were inhibited by the Ca2+ channel blocker verapamil, and exposure of macrophages to BeCl2 had no effect on IP3 concentrations. DNA synthesis, a Ca2+-sensitive function, was enhanced in dividing 1LN cells and induced de novo in quiescent macrophages. Furthermore, BeCl2 enhanced DNA synthesis in the absence of coexposure to the protein kinase C activator phorbol myristate acetate. These data support the hypothesis that beryllium toxicity is in part the result of altered Ca2+ metabolism in mononuclear phagocytes consequent to reversible opening of plasma membrane channels.

Animals↗

PubMed1998

Cyclosporin A inhibits inositol 1,4,5-trisphosphate binding to its receptors and release of calcium from intracellular stores in peritoneal macrophages.

We have studied the effects of the immunosuppressive drug cyclosporin A (CsA) on the generation of inositol 1,4,5-trisphosphate (IP3) and intracellular Ca2+ levels elicited upon ligation of murine macrophage receptors for alpha2-macroglobulin, bradykinin, epidermal growth factor, and platelet-derived growth factor. Preincubation of cells with CsA (500 ng/ml), either alone or with the various ligands, did not inhibit the synthesis of IP3. However, we observed 70-80% inhibition of the binding of [3H]IP3 to IP3 receptors on macrophage membranes isolated from CsA-treated macrophages. Preincubation of macrophages with CsA abolished IP3-mediated release of Ca2+ from intracellular stores and Ca2+ entry from the extracellular medium observed when macrophage receptors were stimulated with ligands in the absence of CsA. Preincubation of macrophages with CsA also significantly inhibited DNA synthesis induced by ligands for all four receptors studied. Thus in macrophages, as in T cells, CsA blocks receptor-activated signal transmission pathways characterized by an initial increase in intracellular Ca2+ concentration. This inhibition appears to result from a drug effect on IP3 receptors.

Animals↗

PubMed1998

Oxidized alpha2-macroglobulin (alpha2M) differentially regulates receptor binding by cytokines/growth factors: implications for tissue injury and repair mechanisms in inflammation.

Alpha2M binds specifically to TNF-alpha, IL-1beta, IL-2, IL-6, IL-8, basic fibroblast growth factor (bFGF), beta-nerve growth factor (beta-NGF), platelet-derived growth factor (PDGF), and TGF-beta. Since many of these cytokines are released along with neutrophil-derived oxidants during acute inflammation, we hypothesize that oxidation alters the ability of alpha2M to bind to these cytokines, resulting in differentially regulated cytokine functions. Using hypochlorite, a neutrophil-derived oxidant, we show that oxidized alpha2M exhibits increased binding to TNF-alpha, IL-2, and IL-6 and decreased binding to beta-NGF, PDGF-BB, TGF-beta1, and TGF-beta2. Hypochlorite oxidation of methylamine-treated alpha2M (alpha2M*), an analogue of the proteinase/alpha2M complex, also results in decreased binding to bFGF, beta-NGF, PDGF-BB, TGF-beta1, and TGF-beta2. Concomitantly, we observed decreased ability to inhibit TGF-beta binding and regulation of cells by oxidized alpha2M and alpha2M*. We then isolated alpha2M from human rheumatoid arthritis synovial fluid and showed that the protein is extensively oxidized and has significantly decreased ability to bind to TGF-beta compared with alpha2M derived from plasma and osteoarthritis synovial fluid. We, therefore, propose that oxidation serves as a switch mechanism that down-regulates the progression of acute inflammation by sequestering TNF-alpha, IL-2, and IL-6, while up-regulating the development of tissue repair processes by releasing bFGF, beta-NGF, PDGF, and TGF-beta from binding to alpha2M.

Animals↗

PubMed1998

Binding of receptor-recognized forms of alpha2-macroglobulin to the alpha2-macroglobulin signaling receptor activates phosphatidylinositol 3-kinase.

Ligation of the alpha2-macroglobulin (alpha2M) signaling receptor by receptor-recognized forms of alpha2M (alpha2M*) initiates mitogenesis secondary to increased intracellular Ca2+. We report here that ligation of the alpha2M signaling receptor also causes a 1. 5-2.5-fold increase in wortmannin-sensitive phosphatidylinositol 3-kinase (PI3K) activity as measured by the quantitation of phosphatidylinositol 3,4,5-trisphosphate (PIP3). PIP3 formation was alpha2M* concentration-dependent with a maximal response at approximately 50 pM ligand concentration. The peak formation of PIP3 occurred at 10 min of incubation. The alpha2M receptor binding fragment mutant K1370R which binds to the alpha2M signaling receptor activating the signaling cascade, increased PIP3 formation by 2-fold. The mutant K1374A, which binds very poorly to the alpha2M signaling receptor, did not cause any increase in PIP3 formation. alpha2M*-induced DNA synthesis was inhibited by wortmannin. 1, 2Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acetoxymethylester a chelator of intracellular Ca2+, drastically reduced alpha2M*-induced increases in PIP3 formation. We conclude that PI3K is involved in alpha2M*-induced mitogenesis in macrophages and intracellular Ca2+ plays a role in PI3K activation.

Androstadienes↗

PubMed1998

Nonproteolytic incorporation of protein ligands into human alpha 2-macroglobulin: implications for the binding mechanism of alpha 2-macroglobulin.

alpha 2-Macroglobulin (alpha 2M) is a complex tetrameric protein of 718 kDa. In native alpha 2M, each of the four subunits contains a thiol ester between the side chains of Cys949 and Gln952. Cleavage of the thiol ester with small nucleophiles destabilizes the native conformation and causes a major conformational change in alpha 2M, which leads to exposure of receptor binding sites and a change in electrophoretic mobility. Recently it has been shown that nucleophilic cleavage of the four thiol esters in alpha 2M is a reversible process with energy requirements dependent on the nucleophile [Grøn, H., Thøgersen, I. B., Enghild, J. J., and Pizzo, S. V. (1996) Biochem. J. 318, 539-545]. The present study is a further investigation of the properties of alpha 2M with cleaved thiol esters and the potential for incorporation of protein ligands at the site of the thiol ester. The thiol ester in alpha 2M was cleaved by NH3. After removal of excess NH3, the alpha 2M derivative was incubated with excess protein ligand (hen egg lysozyme or bovine insulin) at 23, 37, or 50 degreesC, leading to covalent incorporation of the ligands in alpha 2M as analyzed by SDS-PAGE, gel filtration, and centrifugal microfiltration. Receptor binding studies and native pore-limit PAGE confirmed that the alpha 2M derivatives with ligand incorporated remained in the receptor-recognized, "fast" migrating conformation. This is the first demonstration of nonproteolytic, covalent incorporation of protein ligands into receptor-recognized alpha 2M.

Animals↗

PubMed1998

Ligation of the alpha2M signaling receptor with receptor-recognized forms of alpha2-macroglobulin initiates protein and DNA synthesis in macrophages. The effect of intracellular calcium.

We have previously reported that receptor-recognized forms of the proteinase inhibitor alpha2-macroglobulin (alpha2M) bind to a distinct receptor (alpha2MSR), Kd approximately 50-100 pM, activating a signaling cascade, triggering tyrosine phosphorylation of phospholipase Cgamma1, and raising cytosolic pH. We have now studied the effects of alpha2M or a cloned and expressed receptor binding fragment (RBF) on protein and DNA synthesis by macrophages. A nearly linear increase in total protein and DNA synthesis was noted at ligand concentrations up to 100 pM; thereafter, synthesis plateaued. The increase (1.5-2-fold) in protein and DNA synthesis was similar to that observed with known growth factors such as epidermal growth factor and platelet derived growth factor. Mutants of RBF which bind well to alpha2MSR, also caused a similar increase in DNA synthesis. By contrast, mutant K1374R which binds poorly to alpha2MSR demonstrated much less of an effect on DNA synthesis. Chelation of intracellular Ca2+ drastically reduced protein and DNA synthesis induced by RBF or the human growth factors. These studies suggest that activation of native alpha2M, such as would occur during tissue injury, produces a molecule with properties which are similar to growth factors.

Animals↗

PubMed1998

Alterations in calcium metabolism in murine macrophages by the benzene metabolite 1,4-benzoquinone.

Exposure of murine peritoneal macrophages to very low concentrations of 1,4-benzoquinone (BQ) induced immediate increases in intracellular Ca2+ concentrations ([Ca2+]i). Increases in [Ca2+]i were induced by concentrations as low as 5 nM and the response was dose dependent and linear up to 1 microM. The sources of Ca2+ were from both internal inositol triphosphate (IP3)-sensitive and -insensitive sites and from the external medium. BQ did not induce IP3 formation and did not affect binding to its receptors. 1, 4-Hydroquinone had no effect on [Ca2+]i. Catechol did elicit some increases in [Ca2+]i, but did so only at much higher concentrations (5 microM). The action of BQ was almost identical to that of the established Ca2+-ATPase inhibitor thapsigargin except that there were some intracellular stores of Ca2+ released by thapsigargin that were not released by BQ. BQ also was mitogenic for macrophages in conjunction with phorbol myristate acetate. These data suggest that BQ raises [Ca2+]i by inhibition of Ca2+-ATPases, is a comitogen, and does so at concentrations that could be achieved in vivo in the general urban population.

Animals↗

PubMed1998

Ligation of the alpha2M signalling receptor elevates the levels of p21Ras-GTP in macrophages.

Ligation of the alpha2-macroglobulin signalling receptor (alpha2MSR) with alpha2-macroglobulin (alpha2M)-methylamine or a cloned and expressed receptor binding fragment (RBF) stimulates DNA synthesis. To examine the possible role of the Ras pathway in the mitogenic effects observed on ligating alpha2MSR, we studied the formation of p2 Ras-GTP in murine peritoneal macrophages upon treatment with alpha2M-methylamine and RBF, respectively. Both alpha2M-methylamine (50 pM) and RBF (50 pM) stimulated a 2-3-fold increase in the formation of the p21Ras-GTP complex compared with unstimulated cells. p21Ras-GT32P complex formation was both time and RBF concentration dependent and was comparable to p21Ras-GT32P complex formation induced by EGF (200 ng/mL) and platelet derived growth factor (50 mg/mL). Up-regulation of cells with phorbol dibutyrate prior to stimulation with RBF had no effect on p2 Ras-GT32P formation. However, treatment of macrophages with the tyrosine kinase inhibitor genestein drastically reduced RBF-induced formation of the p21 Ras-GT32P complex. Wortmannin, an inhibitor of phosphatidylinositol-3'-kinase (PI3K), had no effect on p21Ras-GT32P complex formation. It is concluded that the mitogenic effects of ligating alpha2MSR are mediated through a Ras-dependent pathway.

Androstadienes↗

PubMed1997

Nickel inhibits binding of alpha2-macroglobulin-methylamine to the low-density lipoprotein receptor-related protein/alpha2-macroglobulin receptor but not the alpha2-macroglobulin signaling receptor.

A previous study demonstrated that activated alpha2-macroglobulin (alpha2M*) binding to the low-density receptor-related protein/alpha2-macroglobulin receptor (LRP/alpha2MR) is blocked by Ni2+ [Hussain, M. M., et al. (1995) Biochemistry 34, 16074-16081]. We now report that the effect of Ni2+ is on a region of the alpha2M molecule upstream of the carboxyl terminal receptor recognition domain. This observation is consistent with previous observations from this laboratory suggesting that alpha2M* binding to LRP/alpha2MR involves a region of the alpha2M molecule immediately upstream of the receptor recognition domain [Enghild, J. J., et al. (1989) Biochemistry 28, 1406-1412]. We further demonstrate that Ni2+ has no effect on the binding of alpha2M* or a cloned and expressed receptor binding fragment (RBF) to the recently described alpha2M signaling receptor as assessed by direct binding and signal transduction studies.

Animals↗

PubMed1997

The binding of receptor-recognized alpha2-macroglobulin to the low density lipoprotein receptor-related protein and the alpha2M signaling receptor is decoupled by oxidation.

Receptor-recognized forms of alpha2-macroglobulin (alpha2M*) bind to two classes of cellular receptors, a high affinity site comprising approximately 1500 sites/cell and a lower affinity site comprising about 60,000 sites/cell. The latter class has been identified as the so-called low density lipoprotein receptor-related protein (LRP). Ligation of receptors distinct from LRP activates cell signaling pathways. Strong circumstantial evidence suggests that the high affinity binding sites are responsible for cell signaling induced by alpha2M*. Using sodium hypochlorite, a powerful oxidant produced by the H2O2-myeloperoxidase-Cl- system, we now demonstrate that binding to the high affinity sites correlates directly with activation of the signaling cascade. Oxidation of alpha2M* using 200 microM hypochlorite completely abolishes its binding to LRP without affecting its ability to activate the macrophage signaling cascade. Scatchard analysis shows binding to a single class of high affinity sites (Kd - 71 +/- 12 pM). Surprisingly, oxidation of native alpha2-macroglobulin (alpha2M) with 125 microM hypochlorite results in the exposure of its receptor-binding site to LRP, but the ligand is unable to induce cell signaling. Scatchard analysis shows binding to a single class of lower affinity sites (Kd - 0.7 +/- 0.15 nM). Oxidation of a cloned and expressed carboxyl-terminal 20-kDa fragment of alpha2M (RBF), which is capable of binding to both LRP and the signaling receptor, results in no significant change in its binding Kd, supporting our earlier finding that the oxidation-sensitive site is predominantly outside of RBF. Attempts to understand the mechanism responsible for the selective exposure of LRP-binding sites in oxidized native alpha2M suggest that partial protein unfolding may be the most likely mechanism. These studies provide strong evidence that the high affinity sites (Kd - 71 pM) are the alpha2M* signaling receptor.

Animals↗

PubMed1997

Evidence for a novel O-linked sialylated trisaccharide on Ser-248 of human plasminogen 2.

Human plasminogen, the inactive precursor of plasmin, exists in two major glycoforms. Plasminogen 1 contains an N-linked oligosaccharide at Asn-289 and an O-linked oligosaccharide at Thr-345. Plasminogen 2 is known to contain only an O-linked oligosaccharide at Thr-345. However, plasminogen 2 displays a further well documented microheterogeneity dependent on the N-acetylneuraminic acid content, which has functional consequences with regard to activation of plasminogen. The proposed structure and number of known oligosaccharide linkages in plasminogen 2 is insufficient to account for this microheterogeneity. In the present study, a combination of trypsin digestion, lectin affinity chromatography, Edman degradation amino acid sequence analysis, carbohydrate composition analysis, and mass spectrometry revealed the existence of a novel site for O-linked glycosylation on plasminogen 2 at Ser-248. Direct evidence for the structure of the carbohydrate was obtained from a combination of lectin affinity chromatography, desialylation experiments, and mass spectrometry analysis. These findings provide a structural basis for some of the observed microheterogeneity, and have implications with regard to the known functional consequences of the extent of sialylation of plasminogen.

Amino Acid Sequence↗

PubMed1997

Up-regulation of the alpha2-macroglobulin signaling receptor on rheumatoid synovial fibroblasts.

In the present study, we demonstrate that the alpha2-macroglobulin (alpha2M) signaling receptor is up-regulated on rheumatoid synovial fibroblasts. In rheumatoid cells, 125I-alpha2M-methylamine bound to two sites; namely, one of high affinity (Kd approximately 52 pM) and the second of lower affinity (Kd approximately 9.7 nM). In normal synovial fibroblasts only one site for 125I-alpha2M-methylamine (Kd approximately 5.36 nM) was present. Receptor-associated protein did not inhibit the binding of alpha2M-methylamine to the high affinity binding sites, but it caused a 70-80% reduction in its binding to low affinity binding sites establishing its identity as the low density lipoprotein receptor-related protein/alpha2M receptor. Binding of alpha2M-methylamine to rheumatoid but not normal synovial fibroblasts caused a rapid rise in inositol 1,4,5-trisphosphate synthesis with a peak reached within 10 s of ligand exposure. Concomitantly, rheumatoid but not normal cells showed a rise in intracellular Ca2+. Pretreatment of rheumatoid cells with Receptor-associated protein or pertussis toxin did not affect the alpha2M-methylamine-induced increase in intracellular Ca2+. These are characteristic properties of ligation by alpha2M-methylamine of the alpha2M signaling receptor but not the lipoprotein receptor-related protein/alpha2M receptor. Binding of alpha2M-methylamine to rheumatoid synovial fibroblasts significantly increased the synthesis of DNA compared with normal synovial fibroblasts treated similarly.

Arthritis, Rheumatoid↗

PubMed1997

Chloroquine, quinine and quinidine inhibit calcium release from macrophage intracellular stores by blocking inositol 1,4,5-trisphosphate binding to its receptor.

The binding of many ligands to cellular receptors induces a signaling cascade which generates inositol 1,4,5-trisphosphate (IP3). IP3 binding to its receptors in various internal compartments causes a rapid Ca2+ efflux into the cytosol. We now demonstrate that chloroquine blocks ligand-induced Ca2+ mobilization without affecting IP3 synthesis. The effect is independent of the ligand employed and occurred with five unrelated ligands; namely, alpha 2-macroglobulin-methylamine, angiotensin II, bradykinin, carbachol, and epidermal growth factor. Chloroquine, quinidine, and quinine, however, block binding of [3H]IP3 to its receptors by 90%, 88%, and 71%, respectively. These observations suggest a previously undetected mechanism by which these agents may in part function as antimalarials.

Animals↗