Antigen-induced Secretion of Histamine and the Phosphorylation of Myosin by Protein Kinase C in Rat Basophilic Leukemia Cells

Abstract. Thrombin stimulates cultured endothelial ceils (EC) to secrete stored von Willebrand factor (vWF), but the signal transduction pathways are poorly defined. Thrombin is known to elevate the concentration of intracellular calcium ([Ca2+]i) and to activate protein kinase C (PKC) in EC. Since both calcium ionophores and phorbol esters release vWF, both second messenger pathways have been postulated to participate in vWF secretion in response to naturally occurring agonists. We find that in intact human EC, vWF secretion stimulated by either thrombin or by a thrombin receptor activating peptide, TR(42-55), can be correlated with agonist-induced elevations of [Ca2+]i. Further evidence implicating calcium in the signal transduction pathway is suggested by the finding that MAPTAM, a cell-permeant calcium chelator, in combination with the extracellular calcium chelator EGTA, can inhibit thrombin-stimulated secretion. In contrast, the observation that staurosporine (a pharmacological inhibitor of PKC) blocks phorbol ester-but not thrombin-stimulated secretion provides evidence against PKC-mediated signal transduction. To examine further the signal transduction pathway initiated by thrombin, we developed novel conditions for minimal permeabilization of EC with saponin (4-8/zg/ml for 5-15 min at 37~ which allow the introduction of small extracellular molecules without the loss of large intraceUular proteins and which retain thrombin-stimulated secretion. These minimally permeabilized cells secrete vWF in response to exogenous calcium, and EGTA blocks thrombin-induced secretion. Moreover, in these cells, thrombin-stimulated secretion is blocked by a calmodulin-binding inhibitory peptide but not by a PKC inhibitory peptide. Taken together, these findings demonstrate that thrombin-stimulated vWF secretion is transduced by a rise in [Ca2+]i and provide the first evidence for the role of calmodulin in this process.

Section: Discussionmentioning

confidence: 92%

Calcium/calmodulin transduces thrombin-stimulated secretion: studies in intact and minimally permeabilized human umbilical vein endothelial cells.

Birch

Pober

Zavoico

et al. 1992

“…YosiN is a diverse superfamily of molecular motors that is currently represented by 12 distinct classes based on sequence homology (40). Myosins of class II (Myosin II) have both a structural and an enzymatic role in such diverse cellular processes as muscle contraction (15), cell division (11), cell locomotion (47,7), and intracellular movements (11,25,30). All myosin II proteins share the same basic molecular structure of a dimer of heavy chains of ~200 kD noncovalently associated with two pairs of light chains of 17 kD and 20 kD.…”

mentioning

confidence: 99%

Xenopus nonmuscle myosin heavy chain isoforms have different subcellular localizations and enzymatic activities.

Kelley

Sellers

Gard

et al. 1996

Self Cite

155

120

Abstract. There are two isoforms of the vertebrate nonmuscle myosin heavy chain, MHC-A and MHC-B, that are encoded by two separate genes. We compared the enzymatic activities as well as the subcellular localizations of these isoforms in Xenopus cells. MHC-A and MHC-B were purified from cells by immunoprecipitation with isoform-specific peptide antibodies followed by elution with their cognate peptides. Using an in vitro motility assay, we found that the velocity of movement of actin filaments by MHC-A was 3.3-fold faster than that by MHC-B. Likewise, the Wma x of the actin-activated Mg 2÷-ATPase activity of MHC-A was 2.6-fold greater than that of MHC-B. Immunofluorescence microscopy demonstrated distinct localizations for MHC-A and MHC-B. In interphase cells, MHC-B was present in the cell cortex and diffusely arranged in the cytoplasm. In highly polarized, rapidly migrating interphase cells, the lamellipodium was dramatically enriched for MHC-B suggesting a possible involvement of MHC-B based contractions in leading edge extension and/or retraction. In contrast, MHC-A was absent from the cell periphery and was arranged in a fibrillar staining pattern in the cytoplasm. The two myosin heavy chain isoforms also had distinct localizations throughout mitosis. During prophase, the MHC-B redistributed to the nuclear membrane, and then resumed its interphase localization by metaphase. MHC-A, while diffuse within the cytoplasm at all stages of mitosis, also localized to the mitotic spindle in two different cultured cell lines as well as in Xenopus blastomeres. During telophase both isoforms colocalized to the contractile ring. The different subcellular localizations of MHC-A and MHC-B, together with the data demonstrating that these myosins have markedly different enzymatic activities, strongly suggests that they have different functions. MYosiN is a diverse superfamily of molecular motors that is currently represented by 12 distinct classes based on sequence homology (40). Myosins of class II (Myosin II) have both a structural and an enzymatic role in such diverse cellular processes as muscle contraction (15), cell division (11), cell locomotion (47, 7), and intracellular movements (11,25,30). All myosin II proteins share the same basic molecular structure of a dimer of heavy chains of ~200 kD noncovalently associated with two pairs of light chains of 17 kD and 20 kD. The myosin heavy chain dimers form two globular amino-terminal heads and a-helical coiled-coil rods. The heads contain an actin-activated ATPase activity while the rods are involved in filament formation. Both the heavy chain and light chain subunits of myosin exist as isoforms. In this paper, we report our studies on the function of isoforms of the heavy chain of nonmuscle myosin II.

“…In contrast to in vitro studies and smooth muscle contraction, in nonmuscle cells there is evidence of functional significance of diphosphorylation of myosin II. Studies examining activation of secretory processes in RBL-2H3 cells (8,48) and platelets (34) suggest that diphosphorylation of myosin light chains correlates with cellular shape change and exocytosis. Likewise in HUVE, although the initial rise in tension is associated with monophosphorylation, development, and maintenance of tension correlates with sustained diphosphorylation.…”

Section: Discussionmentioning

confidence: 99%

Myosin light chain kinase-regulated endothelial cell contraction: the relationship between isometric tension, actin polymerization, and myosin phosphorylation.

Goeckeler

Wysolmerski

1995

412

360

Abstract. The phosphorylation of regulatory myosin light chains by the Ca2÷/calmodulin-dependent enzyme myosin light chain kinase (MLCK) has been shown to be essential and sufficient for initiation of endothelial cell retraction in saponin permeabilized monolayers (Wysolmerski, R. B., and D. Lagunoff. 1990. Proc. Natl. Acad. Sci. . We now report the effects of thrombin stimulation on human umbilical vein endothelial cell (HUVE) actin, myosin II and the functional correlate of the activated actomyosin based contractile system, isometric tension development. Using a newly designed isometric tension apparatus, we recorded quantitative changes in isometric tension from paired monolayers. Thrombin stimulation results in a rapid sustained isometric contraction that increases 2-to 2.5-fold within 5 min and remains elevated for at least 60 min.The phosphorylatable myosin light chains from HUVE were found to exist as two isoforms, differing in their molecular weights and isoelectric points. Resting isometric tension is associated with a basal phosphorylation of 0.54 mol PO4/mol myosin light chain. After thrombin treatment, phosphorylation rapidly increases to 1.61 mol PO4/mol myosin light chain within 60 s and remains elevated for the duration of the experiment. Myosin light chain phosphorylation precedes the development of isometric tension and maximal phosphorylation is maintained during the sustained phase of isometric contraction. Tryptic phosphopeptide maps from both control and thrombin-stimulated cultures resolve both monophosphorylated Ser-19 and diphosphorylated Ser-19/Thr-18 peptides indicative of MLCK activation.Changes in the polymerization of actin and association of myosin II correlate temporally with the phosphorylation of myosin II and development of isometric tension. Activation results in a 57% increase in F-actin content within 90 s and 90% of the soluble myosin II associates with the reorganizing F-actin. Furthermore, the disposition of actin and myosin II undergoes striking reorganization. F-actin initially forms a fine network of filaments that fills the cytoplasm and then reorganizes into prominent stress fibers. Myosin II rapidly forms discrete aggregates associated with the actin network and by 2.5 min assumes a distinct periodic distribution along the stress fibers.NDOTHELIAL cells lining most vessels form a continuous layer that normally constrains proteins as well as formed blood elements to the vascular lumen. The loss of continuity of the endothelial sheet leads to increased permeability and the development of edema (30,47,51,58,71,75). The increase in microvascular permeability by inflammatory and chemical mediators has been correlated with the presence of small gaps between adjacent endothelial cells. Furthermore, mediators that disrupt actin filaments and chelate extracellular calcium have been implicated in changes in macromolecular flux. The factors that may play a role in altering the barrier function of the endothelium leading to edema include: (a) loss of Address all correspondence t...