calyculin-a and herbimycin

Endothelial monolayer hyperpermeability is regulated by a myosin light chain phosphorylation (MLCP)-dependent contractile mechanism. In this study, we tested the role of Src-dependent tyrosine phosphorylation to modulate endothelial contraction and monolayer barrier function with the use of the myosin phosphatase inhibitor calyculin A (CalA) to directly elevate MLCP with the Src family tyrosine kinase inhibitor herbimycin A (HA) in bovine pulmonary artery endothelial cells (EC). CalA stimulated an increase in MLCP, Src kinase activity, an increase in the tyrosine phosphorylation of paxillin and focal adhesion (FA) kinase (p125(FAK)), and monolayer hyperpermeability. Microscopic examination of CalA-treated EC revealed a contractile morphology characterized by peripheral contractile bands of actomyosin filaments and stress fibers linked to phosphotyrosine-containing FAs. These CalA-dependent events were HA sensitive. HA alone stimulated an improvement in monolayer barrier formation by reducing the levels of MLCP and phosphotyrosine-containing proteins and the number of large paracellular holes. These data show that Src kinase plays an important role in regulating monolayer hyperpermeability through adjustments in tyrosine phosphorylation, MLCP, and EC contraction.

Topics: Animals; Benzoquinones; Capillary Permeability; Cattle; Cell Size; Cells, Cultured; Cytoskeletal Proteins; Endothelium, Vascular; Enzyme Inhibitors; Focal Adhesion Protein-Tyrosine Kinases; Lactams, Macrocyclic; Marine Toxins; Myosin Light Chains; Myosin-Light-Chain Phosphatase; Oxazoles; Paxillin; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Protein-Tyrosine Kinases; Quinones; Rifabutin; src-Family Kinases

The neutralization of the polyanionic surface of the podocyte by perfusion of kidneys with polycations, such as protamine sulfate, leads to a retraction of podocyte foot processes and proteinuria. This study investigates the effects of protamine sulfate or anionic, neutral, or cationic dextrans on the cytosolic calcium activity ([Ca2+]i) in podocytes.. [Ca2+]i was measured in single cultured differentiated mouse podocytes with the fluorescence dye fura-2/AM.. Protamine sulfate caused a concentration-dependent and partially reversible increase of [Ca2+]i (EC50 approximately 1.5 micromol/liter). Pretreatment of the cells with heparin (100 U/liter) inhibited the protamine sulfate-mediated increase of [Ca2+]i. Like protamine sulfate, diethylaminoethyl dextran (DEAE-dextran) concentration dependently increased [Ca2+]i in podocytes (EC50 approximately 20 nmol/liter), whereas dextran sulfate or uncharged dextran (both 10 micromol/liter) did not influence [Ca2+]i. A reduction of the extracellular Ca2+ concentration (from 1 mmol/liter to 1 micomol/liter) partially inhibited the protamine sulfate and the DEAE-dextran-induced [Ca2+]i response. Flufenamate (100 micromol/liter) or Gd3+ (10 micromol/liter), which are known to inhibit nonselective ion channels, did not influence the [Ca2+]i increase induced by protamine sulfate. In the presence of thapsigargin (50 nmol/liter), an inhibitor of the endoplasmic reticulum Ca2+-ATPase, both protamine sulfate and DEAE-dextran increased [Ca2+]i.. The data indicate that polycations increase podocyte [Ca2+]i. The increase of [Ca2+]i may be an early event in the pathogenesis of protamine sulfate-mediated retraction of podocyte foot processes.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 3T3 Cells; Animals; Benzoquinones; Calcium Signaling; Cells, Cultured; DEAE-Dextran; Heparin; Kidney Glomerulus; Lactams, Macrocyclic; Marine Toxins; Mice; Muramidase; Oxazoles; Protamines; Quinones; Rifabutin; Staurosporine; Thapsigargin

In resting DDT1MF-2 smooth muscle cells, the cytosolic free Ca2+ concentration ([Ca2+]c) was higher than the free Ca2+ concentration in the nucleus ([Ca2+]n). However, this nucleocytosolic [Ca2+] gradient was reversed by Ca2+ agonists like ATP or, as is shown here, by the epidermal growth factor (EGF). The ATP-induced reversal of the nucleocytosolic [Ca2+] gradient was blocked by stimulation of protein kinase C with phorbol 12-myristate 13-acetate or with the diacylglycerol kinase inhibitor R59949, or by inhibition of the Ser/Thr-specific protein phosphatases-1 and -2A with okadaic acid or calyculin A. Moreover, the magnitude of the ATP-induced reversal of the [Ca2+] gradient diminished during prolonged culture of the cells. The EGF-induced [Ca2+] rise in the cytosol and nucleus was blocked by okadaic acid and by the tyrosine kinase inhibitors herbimycin A and psi-tectorigenin. Our data suggest that the nucleocytosolic [Ca2+] gradient is modulated by (de)phosphorylation processes catalyzed by tyrosine protein kinases, by protein kinase C, and by Ser/Thr protein phosphatases-1 and -2A.

Topics: Adenosine Triphosphate; Animals; Benzoquinones; Calcium; Cell Line; Cell Nucleus; Colforsin; Cricetinae; Cytosol; Diacylglycerol Kinase; Epidermal Growth Factor; Ethers, Cyclic; Homeostasis; Kinetics; Lactams, Macrocyclic; Leiomyosarcoma; Marine Toxins; Mesocricetus; Models, Biological; Muscle, Smooth; Okadaic Acid; Oxazoles; Phosphoproteins; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Piperidines; Protein Kinase C; Protein Kinases; Protein Tyrosine Phosphatases; Quinazolines; Quinazolinones; Quinones; Rifabutin; Tetradecanoylphorbol Acetate; Time Factors; Tumor Cells, Cultured