The α2δ subunit augments functional expression and modifies the pharmacology of CaV1.3 L-type channels

The auxiliary Ca_Vα₂δ-1 subunit is an important component of voltage-gated Ca²⁺ (Ca_V) channel complexes in many tissues and of great interest as a drug target. Nevertheless, its exact role in specific cell functions is still unknown. This is particularly important in the case of the neuronal L-type Ca_V channels where these proteins play a key role in the secretion of neurotransmitters and hormones, gene expression, and the activation of other ion channels. Therefore, using a combined approach of patch-clamp recordings and molecular biology, we studied the role of the Ca_Vα₂δ-1 subunit on the functional expression and the pharmacology of recombinant L-type Ca_V1.3 channels in HEK-293 cells. Co-expression of Ca_Vα₂δ-1 significantly increased macroscopic currents and conferred the Ca_V1.3α₁/Ca_Vβ₃ channels sensitivity to the antiepileptic/analgesic drugs gabapentin and AdGABA. In contrast, Ca_Vα₂δ-1 subunits harboring point mutations in N-glycosylation consensus sequences or the proteolytic site as well as in conserved cysteines in the transmembrane δ domain of the protein, reduced functionality in terms of enhancement of Ca_V1.3α₁/Ca_Vβ₃ currents. In addition, co-expression of the δ domain drastically inhibited macroscopic currents through recombinant Ca_V1.3 channels possibly by affecting channel synthesis. Together these results provide several lines of evidence that the Ca_Vα₂δ-1 auxiliary subunit may interact with Ca_V1.3 channels and regulate their functional expression.     

Integrin αvβ3, metalloproteinases, and sphingomyelinase-2 mediate urokinase mitogenic effect

Plasminogen activators are implicated in the pathogenesis of several diseases such as inflammatory diseases and cancer. Beside their serine-protease activity, these agents trigger signaling pathways involved in cell migration, adhesion and proliferation. We previously reported a role for the sphingolipid pathway in the mitogenic effect of plasminogen activators, but the signaling mechanisms involved in neutral sphingomyelinase-2 (NSMase-2) activation (the first step of the sphingolipid pathway) are poorly known. This study was carried out to investigate how urokinase plasminogen activator (uPA) activates NSMase-2. We report that uPA, as well as its catalytically inactive N-amino fragment ATF, triggers the sequential activation of MMP-2, NSMase-2 and ERK1/2 in ECV304 cells that are required for uPA-induced ECV304 proliferation, as assessed by the inhibitory effect of Marimastat (a MMP inhibitor), MMP-2-specific siRNA, MMP-2 defect, and NSMase-specific siRNA. Moreover, upon uPA stimulation, uPAR, MT1-MMP, MMP-2 and NSMase-2 interacted with integrin α_vβ₃, evidenced by co-immunoprecipitation and immunocytochemistry experiments. Moreover, the α_vβ₃ blocking antibody inhibited the uPA-triggered MMPs/uPAR/integrin α_vβ₃ interaction, NSMase-2 activation, Ki67 expression and DNA synthesis in ECV304. In conclusion, uPA triggers interaction between integrin α_vβ₃, uPAR and MMPs that leads to NSMase-2 and ERK1/2 activation and cell proliferation. These findings highlight a new signaling mechanism for uPA, and suggest that, upon uPA stimulation, uPAR, MMPs, integrin α_vβ₃ and NSMase-2 form a signaling complex that take part in mitogenic signaling in ECV304 cells.     

Activation of integrin alpha(V)beta(3) regulates cell adhesion and migration to bone sialoprotein

alpha(V)beta(3), a broadly distributed member of the integrin family of adhesion receptors, has been implicated in a variety of physiological and pathophysiological events, including control of bone density, angiogenesis, apoptosis, tumor growth, and metastasis. Recently, it has been shown that activation of alpha(V)beta(3), its transition from a low- to a high-affinity/avidity state, influences its recognition of certain ligands. Bone sialoprotein (BSP) is recognized as an important ligand for alpha(V)beta(3) in processes ranging from bone formation to the homing of metastatic tumor cells. Here, the influence of alpha(V)beta(3) activation on the adhesion and migration of relevant cells to BSP has been examined. Stimulation of lymphoblastoid, osteoblastoid, and human umbilical vein endothelial cells (HUVEC) with PMA or Mn(2+) markedly enhanced alpha(V)beta(3)-dependent adhesion to BSP. alpha(V)beta(3)-mediated migration of HUVEC or osteoblastic cells to BSP was substantially enhanced by stimulation, demonstrating that alpha(V)beta(3) activation enhances both adhesive and migratory responses. However, adhesion and/or migration of certain tumor cell lines, including M21 melanoma and MDA MB435 and SKBR3 breast carcinoma cell lines, to BSP was constitutively high and was not augmented by alpha(V)beta(3)-activating stimuli. Inhibitors of the intracellular signaling molecules, phosphatidylinositol 3-kinase with wortmannin, hsp90-dependent kinases with geldanamycin, and calpain with calpeptin, but not MAPKK with PD98059, reduced the high spontaneous adhesion and migration of the M21 cells to BSP, consistent with the constitutive activation of the receptor on these tumor cells. These results indicate that the activation state of alpha(V)beta(3) can regulate cell migration and adhesion to BSP and, by extension, to other ligands of this receptor. The constitutive activation of alpha(V)beta(3) on neoplastic cells may contribute to tumor growth and metastatic potential.     

β1 And β3 integrins have different roles in the adhesion and migration of vascular smooth muscle cells on extracellular matrix

Extracellular matrix receptors on ductus arteriosus smooth muscle cells (SMC) must enable the cells to migrate through both interstitial and basement membrane matrices to form intimal mounds during postnatal ductus closure. We examined the role of β₁ and β₃ integrin receptors on SMC adhesion and migration. Using a new assay to measure cell migration, we found that lamb ductus arteriosus SMC attach to and migrate over surfaces coated with fibronectin (FN), laminin (LN), vitronectin (VN), and collagens I (I) and IV (IV). Blocking antibodies, specific to different integrin complexes, showed that SMC adhesion to FN, LN, I, and IV depended exclusively on functioning β₁ integrins with little, if any, contribution by the α_vβ₃ integrin; on the other hand, cell migration over these substrates depended to a large extent on the α_vβ₃ receptor. Immunofluorescent staining demonstrated that during the early phase of SMC migration, the β₁ integrins organized rapidly into focal plaques that, with time, gradually covered the cell's basal surface; on the other hand, the β₃ receptor remained concentrated at all times at the cell's margins. Ligand affinity chromatography and immunoprecipitation techniques identified a unique series of β₁ integrins binding to each matrix component: FN (α₅β₁, α₃β₁, α_vβ₁), LN (α₁β₁, α₇β₁), VN (α_vβ₁), I (α₁β₁, α₂β₁), and IV (α₁β₁). In contrast, the β₃ integrin, α_vβ₃, bound to all the substrates tested: FN, LN, VN, I, and IV. The results indicate that β₁ and β₃ integrins may play different roles in attachment and migration as SMC move through the vascular extracellular matrix to produce obliteration of the ductus arteriosus lumen.     

Activation of αVβ3 on Vascular Cells Controls Recognition of Prothrombin

Regulation of vascular homeostasis depends upon collaboration between cells of the vessel wall and blood coagulation system. A direct interaction between integrin α_Vβ₃ on endothelial cells and smooth muscle cells and prothrombin, the pivotal proenzyme of the blood coagulation system, is demonstrated and activation of the integrin is required for receptor engagement. Evidence that prothrombin is a ligand for α_Vβ₃ on these cells include: (a) prothrombin binds to purified α_Vβ₃ via a RGD recognition specificity; (b) prothrombin supports α_Vβ₃-mediated adhesion of stimulated endothelial cells and smooth muscle cells; and (c) endothelial cells, either in suspension and in a monolayer, recognize soluble prothrombin via α_Vβ₃. α_Vβ₃-mediated cell adhesion to prothrombin, but not to fibrinogen, required activation of the receptor. Thus, the functionality of the α_Vβ₃ receptor is ligand defined, and prothrombin and fibrinogen represent activation- dependent and activation-independent ligands. Activation of α_Vβ₃ could be induced not only by model agonists, PMA and Mn²⁺, but also by a physiologically relevant agonist, ADP. Inhibition of protein kinase C and calpain prevented activation of α_Vβ₃ on vascular cells, suggesting that these molecules are involved in the inside-out signaling events that activate the integrin. The capacity of α_Vβ₃ to interact with prothrombin may play a significant role in the maintenance of hemostasis; and, at a general level, ligand selection by α_Vβ₃ may be controlled by the activation state of this integrin.     

E-Selectin Mediated Adhesion and Migration of Endothelial Colony Forming Cells Is Enhanced by SDF-1α/CXCR4

Objective

Endothelial-colony forming cells (ECFCs) can be readily expanded from human umbilical cord blood and can facilitate repair of endothelial injury. E-selectin and SDF-1α are produced following endothelial injury and can regulate endothelial progenitor homing. Mechanisms of vascular repair specific to the mode of injury have not been well described in homogenous cell populations such as ECFCs and are needed for development of more effective vascular repair strategies.

Methods and Results

Lipopolysaccharide (LPS)-induced endotoxic injury to mature human umbilical vein endothelial cells (HUVEC) was compared with hypoxic and radiation injury. E-selectin expression in HUVEC cells is markedly increased (208-fold) following LPS-induced injury and facilitates increased ECFC adhesion and migration function in vitro. SDF-1α expression remains unchanged in LPS-treated HUVEC cells but increases more than 2 fold in fibroblasts undergoing similar endotoxic injury. SDF-1α induces expression of E-selectin ligands on ECFCs and facilitates greater E-selectin-mediated adhesion and migration of ECFCs in a CXCR4-dependent manner. Induction of E-selectin expression in HUVECs following hypoxic or radiation injury is negligible, however, while SDF-1α is increased markedly following hypoxia, highlighting injury-specific synergism between mediators of vascular repair.

Conclusion

E-selectin mediates adhesion and migration of ECFCs following endotoxic endothelial injury. SDF-1α augments E-selectin mediated ECFC adhesion and migration in a CXCR4-dependent manner.     

Contrasting effects of interferon γ and interleukin 4 on responses of human vascular endothelial cells to tumour necrosis factor α

Tumour necrosis factor α (TNF-α) and interleukin 4 (IL-4) selectively synergise in inducing expression of the mononuclear cell adhesion receptor VCAM-1 (vascular cell adhesion molecule-1) on human umbilical vein endothelialcells (HUVEC), which results in increased adhesiveness of HUVEC for T lymphocytes. This process may be crucial for adherence of circulating lymphocytes prior to their passage from the blood into inflammatory tissues. IL-4 also amplifies production of interleukin 6 (IL-6) and monocyte chemotactic protein-(MCP-1) from TNF-α-activated HUVEC. In the present study we demonstrate that IL-4 enhances production of granulocyte-macrophage colon-stimulating factor (GM-CSF) from TNF-α-stimulated HUVEC. Moreover, using cultured adult saphenous vein and umbilical artery endothelial cells, we show identical effects of IL-4 on TNF-α-induced responses to those observed with endothelial cells of foetal origin. Additionaly, we report here that TNF-α and interferon γ (IFN-γ) synergise in the induction of both the lymphocyte adhesion receptor VCAM-1, and the TNF-α-inducible neutrophil adhesion receptor intercellular adhesion molecule-1, on all three endothelial cell types studied. In contrast, we found that GM-CSF secretion by endothelial cells treated with IFN-γ plus TNF-α was markedly decreased when compared to the response by TNF-α alone. These results suggest that the combined actions of several cytokines, acting sequentially or in concert, may exert differential effects on activation and accumulation of circulating lymphocytes at sites of inflammation.     

The angiogenic response is dictated by β3 integrin on bone marrow–derived cells

Angiogenesis is dependent on the coordinated action of numerous cell types. A key adhesion molecule expressed by these cells is the α_vβ₃ integrin. Here, we show that although this receptor is present on most vascular and blood cells, the key regulatory function in tumor and wound angiogenesis is performed by β₃ integrin on bone marrow–derived cells (BMDCs) recruited to sites of neovascularization. Using knockin mice expressing functionally stunted β₃ integrin, we show that bone marrow transplantation rescues impaired angiogenesis in these mice by normalizing BMDC recruitment. We demonstrate that α_vβ₃ integrin enhances BMDC recruitment and retention at angiogenic sites by mediating cellular adhesion and transmigration of BMDCs through the endothelial monolayer but not their release from the bone niche. Thus, β₃ integrin has the potential to control processes such as tumor growth and wound healing by regulating BMDC recruitment to sites undergoing pathological and adaptive angiogenesis.     

Molecular determinants of desensitization and assembly of the chimeric GABAA receptor subunits (α1/γ2) and (γ2/α1) in combinations with β2 and γ2

Two γ-aminobutyric acid_A (GABA_A) receptor chimeras were designed in order to elucidate the structural requirements for GABA_A receptor desensitization and assembly. The (α1/γ2) and (γ2/α1) chimeric subunits representing the extracellular N-terminal domain of α1 or γ2 and the remainder of the γ2 or α1 subunits, respectively, were expressed with β2 and β2γ2 in Spodoptera frugiperda (Sf-9) cells using the baculovirus expression system. The (α1/γ2)β2 and (α1/γ2)β2γ2 but not the (γ2/α1)β2 and (γ2/α1)β2γ2 subunit combinations formed functional receptor complexes as shown by whole-cell patch–clamp recordings and [³H]muscimol and [³H]flunitrazepam binding. Moreover, the surface immunofluorescence staining of Sf-9 cells expressing the (α1/γ2)-containing receptors was pronounced, as opposed to the staining of the (γ2/α1)-containing receptors, which was only slightly higher than background. To explain this, the (α1/γ2) and (γ2/α1) chimeras may act like α1 and γ2 subunits, respectively, indicating that the extracellular N-terminal segment is important for assembly. However, the (α1/γ2) chimeric subunit had characteristics different from the α1 subunit, since the (α1/γ2) chimera gave rise to no desensitization after GABA stimulation in whole-cell patch–clamp recordings, which was independent of whether the chimera was expressed in combination with β2 or β2γ2. Surprisingly, the (α1/γ2)(γ2/α1)β2 subunit combination did desensitize, indicating that the C-terminal segment of the α1 subunit may be important for desensitization. Moreover, desensitization was observed for the (α1/γ2)β2γ2 receptor with respect to the direct activation by pentobarbital. This suggests differences in the mechanism of channel activation for pentobarbital and GABA.     

Enhanced Expression of αV Integrin Subunit and Osteopontin during Differentiation of HL-60 Cells along the Monocytic Pathway

HL-60 cells, a promyelocytic leukemic cell line, provide a good model for studying the role of adhesion molecules and associated receptors involved in cell differentiation. When exposed to factors such as phorbol esters, these cells grown in suspension differentiate into monocytes and adhere to tissue culture dishes. In this study we showed that HL-60 cells exposed to phorbol esters express osteopontin (OPN), a cell adhesion molecule linked with osteoclast function. Moreover, the timed expression of OPN, in phorbol ester treated cells, was linked to increased cell adhesion. Subsequent to the expression of OPN, an increase in mRNA levels for α_V integrin subunit was observed. The α_Vβ₃ integrin, a cell surface receptor found in high concentrations in osteoclasts, is considered to be a receptor for OPN. Furthermore, during differentiation we detected an increase in two cell surface markers specific for osteoclasts, 75B and 121F. This is the first report to demonstrate expression of OPN during differentiation of HL-60 cells, indicating that HL-60 cells can be used as a tool to enhance our understanding as to the role of OPN in cell differentiation.     

Requirement for Tyrosine Kinase-ERK1/2 Signaling in α1β1 Integrin-Mediated Collagen Matrix Remodeling by Rat Mesangial Cells

Abnormal mesangial extracellular matrix remodeling by mesangial cells (MCs) is the hallmark of progressive glomerulonephritis (GN). We recently showed, using a type I collagen gel contraction assay, that α1β1 integrin-dependent MC adhesion and migration are necessary cell behaviors for collagen matrix remodeling. To further determine the mechanism of α1β1 integrin-mediated collagen remodeling, we studied the signaling pathways of MCs that participate in the regulation of collagen gel contraction. Immunoprecipitation and phosphotyrosine detection revealed that gel contraction is associated with the enhanced activity and phosphorylation of ERK1/2 by MCs. The tyrosine kinase inhibitors herbimycin and genistein inhibited collagen gel contraction dose dependently. Furthermore, targeting ERK1/2 activity with a MEK inhibitor, PD98059, and antisense ERK1/2 hindered gel contraction in a dose-dependent manner. Similar inhibitory effects on gel contraction and ERK1/2 phosphorylation were observed when MC-mediated gel contraction was performed in the presence of function-blocking anti-α1 or anti-β1 integrin antibodies. However, cell adhesion and migration assays indicated that PD98059 and antisense ERK1/2 blocked α1β1 integrin-dependent MC migration, but did not interfere with collagen adhesion, although there was a marked decrease in ERK1/2 phosphorylation and ERK1/2 protein expression in cell adhesion on type I collagen. None of the above could affect membrane expression of α1β1 integrin. These results suggested that ERK1/2 activation is critical for the α1β1 integrin-dependent MC migration necessary for collagen matrix reorganization. We therefore conclude that ERK1/2 may serve as a possible target for pharmacological inhibition of pathological collagen matrix formation in GN.     

Integrin Cross Talk: Activation of Lymphocyte Function-associated Antigen-1 on Human T Cells Alters α4β1- and α5β1-mediated Function

A regulated order of adhesion events directs leukocytes from the vascular compartment into injured tissues in response to inflammatory stimuli. We show that on human T cells, the interaction of the β2 integrin leucocyte function–associated antigen-1 (LFA-1) with its ligand intercellular adhesion molecule-1 (ICAM-1) will decrease adhesion mediated by α4β1 and, to a lesser extent, α5β1. Similar inhibition is also seen when T cells are exposed to mAb 24, which stabilizes LFA-1 in an active state after triggering integrin function through divalent cation Mg²⁺, PdBu, or T cell receptor/ CD3 complex (TCR/CD3) cross-linking. Such cross talk decreases α4β1 integrin–mediated binding of T cells to fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In contrast, ligand occupancy or prolonged activation of β1 integrin has no effect on LFA-1 adhesion to ICAM-1. We also show that T cell migration across fibronectin, unlike adhesion, is mediated solely by α5β1, and is increased when the α4β1-mediated component of fibronectin adhesion is decreased either by cross talk or the use of α4-blocking mAb. The ability of mAb 24 Fab′ fragments to induce cross talk without cross-linking LFA-1 suggests signal transduction through the active integrin. These data provide the first direct evidence for cross talk between LFA-1 and β1 integrins on T cells. Together, these findings imply that activation of LFA-1 on the extravasating T cell will decrease the binding to VCAM-1 while enhancing the subsequent migration on fibronectin. This sequence of events provides a further level of complexity to the coordination of T cell integrins, whose sequential but overlapping roles are essential for transmigration.     

A Phosphoinositide 3-Kinase/Phospholipase Cgamma1 Pathway Regulates Fibroblast Growth Factor-Induced Capillary Tube Formation

Background

The fibroblast growth factors (FGFs) are key regulators of embryonic development, tissue homeostasis and tumour angiogenesis. Binding of FGFs to their receptor(s) results in activation of several intracellular signalling cascades including phosphoinositide 3-kinase (PI3K) and phospholipase C (PLC)γ1. Here we investigated the basic FGF (FGF-2)-mediated activation of these enzymes in human umbilical vein endothelial cells (HUVECs) and defined their role in FGF-2-dependent cellular functions.

Methodology/Principal Findings

We show that FGF-2 activates PLCγ1 in HUVECs measured by analysis of total inositol phosphates production upon metabolic labelling of cells and intracellular calcium increase. We further demonstrate that FGF-2 activates PI3K, assessed by analysing accumulation of its lipid product phosphatidylinositol-3,4,5-P₃ using TLC and confocal microscopy analysis. PI3K activity is required for FGF-2-induced PLCγ1 activation and the PI3K/PLCγ1 pathway is involved in FGF-2-dependent cell migration, determined using Transwell assay, and in FGF-2-induced capillary tube formation (tubulogenesis assays in vitro). Finally we show that PI3K-dependent PLCγ1 activation regulates FGF-2-mediated phosphorylation of Akt at its residue Ser473, determined by Western blotting analysis. This occurs through protein kinase C (PKC)α activation since dowregulation of PKCα expression using specific siRNA or blockade of its activity using chemical inhibition affects the FGF-2-dependent Ser473 Akt phosphorylation. Furthermore inhibition of PKCα blocks FGF-2-dependent cell migration.

Conclusion/Significance

These data elucidate the role of PLCγ1 in FGF-2 signalling in HUVECs demonstrating its key role in FGF-2-dependent tubulogenesis. Furthermore these data unveil a novel role for PLCγ1 as a mediator of PI3K-dependent Akt activation and as a novel key regulator of different Akt-dependent processes.     

TGF-β3-Induced Chondroitin Sulphate Proteoglycan Mediates Palatal Shelf Adhesion

In mammals, the adhesion and fusion of the palatal shelves are essential mechanisms in the development of the secondary palate. Failure of any of these processes leads to the formation of cleft palate. The mechanisms underlying palatal shelf adhesion are poorly understood, although the presence of filopodia on the apical surfaces of the superficial medial edge epithelial (MEE) cells seems to play an important role in the adhesion of the opposing MEE. We demonstrate here the appearance of chondroitin sulphate proteoglycan (CSPG) on the apical surface of MEE cells only immediately prior to contact between the palatal shelves. This apical CSPG has a functional role in palatal shelf adhesion, as either the alteration of CSPG synthesis by β-d-Xyloside or its specific digestion by chondroitinase AC strikingly alters the in vitro adhesion of palatal shelves. We also demonstrate the absence of this apical CSPG in the clefted palates of transforming growth factor beta 3 (TGF-β₃) null mutant mice, and its induction, together with palatal shelf adhesion, when TGF-β₃ is added to TGF-β₃ null mutant palatal shelves in culture. When chick palatal shelves (that do not adherein vivo nor express TGF-β₃, nor CSPG in the MEE) are cultured in vitro, they do not express CSPG and partially adhere, but when TGF-β₃ is added to the media, they express CSPG and their adhesion increases strikingly. We therefore conclude that the expression of CSPG on the apical surface of MEE cells is a key factor in palatal shelf adhesion and that this expression is regulated by TGF-β₃.     

Cardiovascular actions of prostaglandins F2α; 15-me-F2α; F1α; F2β and F1β in the cat

Prostaglandin F_2α (5μg/kg, i.v.) causes an increase in pulmonary arterial pressure, decrease in systemic arterial pressure, and reflex bradycardia in the anesthetized cat. The same dose of the 15-methyl analogue of PGF_2α produces the same triad of effects but of greater magnitude and duration. Although prostaglandins F_1α, F_2β and F_1β also cause the same cardiovascular effects as F_2α, there is a decrease in potency for all parameters measured, with PGF_2α>PGF_1α>PGF_2β>PGF_1β. When compared to the actions of PGF_2α in producing an increase in pulmonary arterial pressure, PGs F_1α, F_2β and F_1β were less potent by approximately 10, 100, and 1000 fold respectively.     

Osteopontin-Stimulated Vascular Smooth Muscle Cell Migration Is Mediated by β3 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 EC₅₀ 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 β₃. In contrast, polyclonal antiserum recognizing the rat integrin β₁ 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_vβ₃, could play a role in the cells' response to vascular injury and especially neointima formation.     

Release of 6-keto-prostaglandin F1α and thromboxane B2 from mouse peritoneal macrophages during their adhesion and spreading on a glass surface

The release of 6-keto-prostaglandin F_1α (6KF_1α)_and of thromboxane B₂ (TXB₂) from cells were investigated using mouse peritoneal exudate cells (PECs) and non-cultured peritoneal macrophages. They were prepared by adhesion to glass dishes and incubated for 1 hr at 37°C in 5% Co₂ in air. Both the percentage of spreading macrophages and the release of 6KF_1α and TXb₂ increased in proportion to the incubation time. 6KF_1α and TXB₂ were released from the macrophages, not from the non-adherent cells. When PECs were incubated in silicon-coated glass dishes, the spreading of macrophages was hardly detected and lower amounts of 6KF_1α and TXB₂ were released from these cells compared with cells incubated in non-treated glass dishes. These findings suggest that adhesion with the correlated spreading of macrophages on glass dishes serve as a considerable physical factor for the release of 6KF_1α and TXB₂.