Regulation of cell volume and intracellular pH in hyposmotically swollen rat osteosarcoma cells

1995 ◽  
Vol 73 (7-8) ◽  
pp. 535-544 ◽  
Author(s):  
C. Lo ◽  
J. Ferrier ◽  
H. C. Tenenbaum ◽  
C. A. G. McCulloch
Keyword(s):  
Cell Volume ◽  
Intracellular Ph ◽  
Binding Proteins ◽  
Kinase Inhibitor ◽  
Regulatory Volume Decrease ◽  
Cell Swelling ◽  
Osteosarcoma Cells ◽  
Extracellular Medium ◽  
Gtp Binding Proteins ◽  
Gtp Binding

The maintenance of cell volume involves transduction of a volume-sensing signal into effectors of volume-regulatory transporters. After exposure to anisotonic conditions, cells undergo compensatory volume changes that are mediated by active transport and passive movement of ions and solutes. Intracellular pH (pHi) homeostasis may be compromised during these processes. We have studied pHi and some of the signal transduction mechanisms involved in the regulatory volume decrease (RVD) that occurs after exposure to hypoosmolar conditions in rat osteosarcoma cells, ROS 17/2.8. Cells were loaded with BCECF; pHi and cell volume were estimated by dual excitation ratio fluorimetry. Swelling of cells in 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES) buffered hypotonic medium induced a rapid cell swelling followed by an incomplete RVD of ~30% in suspended (i.e., round) cells and ~60% in attached (i.e., spread) cells that was independent of subpassage number. RVD was inhibited by ouabain, valinomycin, and high external [K+], all of which should reduce the cell membrane electrochemical gradient for K+. Inhibition of RVD was induced also by decreasing intracellular [Ca2+] with B APTA–AM and by depletion of Cl−, indicating the role of calcium-regulated K+ and Cl− efflux during RVD. Depolymerization of actin filaments by cytochalasin D prolonged the RVD three-fold and nonspecific activation of GTP-binding proteins up-regulated RVD. In attached cells the hypoosmolar-induced swelling caused a large reduction in pHi (~0.7 units), which was sustained as long as cells were in hypoosmotic medium. The reduction of pHi induced by cell swelling was inhibited by Na+-free extracellular medium, ouabain, the tyrosine kinase inhibitor genistein, and to a lesser extent by Cl−-free medium. However, amiloride failed to inhibit the hypoosmolar-induced reduction of pHi. Collectively these data indicate that RVD of ROS 17/2.8 cells in HEPES-buffered medium is dependent on conductive efflux of K+ and Cl− that is regulated by cell shape, actin, and GTP-binding proteins. The sustained inhibition of pHi homeostasis induced by cell swelling may reflect the existence of cell volume sensing mechanisms that operate through tyrosine kinases to regulate pHi.Key words: cell volume, pH, osteoblast, G proteins, actin.

Shedding of Procoagulant Microparticles from Platelets through Integrin Outside-In Signaling Involving WASP and the Actin Cytoskeleton.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1529-1529
Author(s):  
Sandra Cauwenberghs ◽  
Marion A.H. Feijge ◽  
Alan G.H. Harper ◽  
Stewart O. Sage ◽  
Elisabeth van Pampus ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Cellular Response ◽  
Binding Proteins ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Procoagulant Effect ◽  
Phosphoinositide 3 Kinase ◽  
Calpain Inhibition

Abstract Platelet activation by potent calcium-mobilizing agents, like collagen and thrombin or ionomycin, results in Ca2+- and calpain-dependent shedding of microparticles (MP) that are active in coagulation. Here, we describe and characterize a different mechanism of MP formation by platelets under storage in the absence of agonists. This shedding relies on integrin αIIbβ3 activity, since clinically used integrin blockers, like tirofiban or abciximab, reduce storage-induced MP shedding by 83±5.7% (p=0.006); inhibition of intracellular integrin signaling via Src kinase (PP1) or phosphoinositide 3-kinase (PI-3 kinase, wortmannin) inhibits this process by 52.3±14.3% (p=0.035) and 39±7% (p=0.005), respectively. Calpain inhibition suppresses MP formation by only 35±3.8% (p=0.003), while calpain activity is also not increased during storage. Levels of cytosolic Ca2+ concentration remain at 25–30 nM in the stored platelets. Treatment of platelets with recombinant WASP (Wiskott-Aldrich Syndrome Protein) peptide, activating the Arp2/3 complex and promoting actin nucleation, affects storage-induced MP shedding (p=0.001). Rho and Rac1, belonging to the family of Rho GTP-binding proteins, are involved in actin cytoskeleton regulation and platelet spreading. However, these GTP-binding proteins do not seem to be involved in MP shedding, as treatment of platelets with recombinant Rac1 peptide or with Rho kinase inhibitor is ineffective, as in the absence of these GTP-binding proteins. Furthermore, incubation of platelets with cytochalasin D, latrunculin B or jasplakinolide, which compounds affect actin filament polymerization, increases the amount of procoagulant MP (p<0.032). Interestingly, this effect is greatly reverted in the presence of integrin blockers or wortmannin. We determine that coagulation-active MP are abundantly present in stored platelet preparations that are used for transfusion purposes. The contribution of these MP to the coagulation process was studied after transfusion of these preparations to thrombocytopenic patients. Thrombin generation measurements indicate that the transfused MP initiate up to 25 nM concentrations of thrombin, which potentiate the procoagulant effect of platelets in these patients. Taken together, we conclude that the process of integrin-mediated MP shedding from apparently resting platelets is a uniquely regulated cellular response that operates independently of Ca2+ elevation and calpain activation, and contributes to coagulant activity in a clinically relevant way. It involves αIIbβ3 outside-in signaling, leading to WASP-mediated destabilization of the actin cytoskeleton, but independently of Rho and Rac1.

scholarly journals Rho family GTP binding proteins are involved in the regulatory volume decrease process in NIH3T3 mouse fibroblasts

2002 ◽  
Vol 541 (3) ◽  
pp. 779-796 ◽  
Author(s):  
Stine F. Pedersen ◽  
Kristine H. Beisner ◽  
Charlotte Hougaard ◽  
Berthe M. Willumsen ◽  
Ian H. Lambert ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Regulatory Volume Decrease ◽  
Mouse Fibroblasts ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Rho Family ◽  
Volume Decrease

Small GTP-binding Protein RalA Associates with Weibel-Palade Bodies in Endothelial Cells

1999 ◽  
Vol 82 (09) ◽  
pp. 1177-1181 ◽  
Author(s):  
Hubert de Leeuw ◽  
Pauline Wijers-Koster ◽  
Jan van Mourik ◽  
Jan Voorberg
Keyword(s):  
Endothelial Cells ◽  
Binding Proteins ◽  
Binding Protein ◽  
Control Release ◽  
Small Gtpase ◽  
Gtp Binding Protein ◽  
Two Dimensional Gel Electrophoresis ◽  
Gtp Binding Proteins ◽  
Dense Granules ◽  
Gtp Binding

SummaryIn endothelial cells von Willebrand factor (vWF) and P-selectin are stored in dense granules, so-called Weibel-Palade bodies. Upon stimulation of endothelial cells with a variety of agents including thrombin, these organelles fuse with the plasma membrane and release their content. Small GTP-binding proteins have been shown to control release from intracellular storage pools in a number of cells. In this study we have investigated whether small GTP-binding proteins are associated with Weibel-Palade bodies. We isolated Weibel-Palade bodies by centrifugation on two consecutive density gradients of Percoll. The dense fraction in which these subcellular organelles were highly enriched, was analysed by SDS-PAGE followed by GTP overlay. A distinct band with an apparent molecular weight of 28,000 was observed. Two-dimensional gel electrophoresis followed by GTP overlay revealed the presence of a single small GTP-binding protein with an isoelectric point of 7.1. A monoclonal antibody directed against RalA showed reactivity with the small GTP-binding protein present in subcellular fractions that contain Weibel-Palade bodies. The small GTPase RalA was previously identified on dense granules of platelets and on synaptic vesicles in nerve terminals. Our observations suggest that RalA serves a role in regulated exocytosis of Weibel-Palade bodies in endothelial cells.

Rap1b Association with the Platelet Cytoskeleton Occurs in the Absence of Glycoproteins IIb/IIIa

1998 ◽  
Vol 79 (04) ◽  
pp. 832-836 ◽  
Author(s):  
Thomas Fischer ◽  
Christina Duffy ◽  
Gilbert White
Keyword(s):  
Molecular Weight ◽  
Divalent Cation ◽  
Binding Proteins ◽  
Binding Protein ◽  
Platelet Membrane ◽  
Low Molecular Weight ◽  
Membrane Glycoproteins ◽  
Gtp Binding Protein ◽  
Gtp Binding Proteins ◽  
Gtp Binding

SummaryPlatelet membrane glycoproteins (GP) IIb/IIIa and rap1b, a 21 kDa GTP binding protein, associate with the triton-insoluble, activation-dependent platelet cytoskeleton with similar rates and divalent cation requirement. To examine the possibility that GPIIb/IIIa was required for rap1b association with the cytoskeleton, experiments were performed to determine if the two proteins were linked under various conditions. Chromatography of lysates from resting platelets on Sephacryl S-300 showed that GPIIb/IIIa and rap1b were well separated and distinct proteins. Immunoprecipitation of GPIIb/IIIa from lysates of resting platelets did not produce rap1b or other low molecular weight GTP binding proteins and immunoprecipitation of rap1b from lysates of resting platelets did not produce GPIIb/IIIa. Finally, rap1b was associated with the activation-dependent cytoskeleton of platelets from a patient with Glanzmann’s thrombasthenia who lacks surface expressed glycoproteins IIb and IIIa. Based on these findings, we conclude that no association between GPIIb/IIIa and rap1b is found in resting platelets and that rap1b association with the activation-dependent cytoskeleton is at least partly independent of GPIIb/IIIa.

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