R.I. Sha'afi

Vascular endothelial cell adherens junction assembly and morphogenesis induced by sphingosine-1-phosphate.

Vascular endothelial cells undergo morphogenesis into capillary networks in response to angiogenic factors. We show here that sphingosine-1-phosphate (SPP), a platelet-derived bioactive lipid, activates the EDG-1 and -3 subtypes of G protein-coupled receptors on endothelial cells to regulate angiogenesis. SPP induces the Gi/mitogen-activated protein kinase/cell survival pathway and the small GTPase Rho- and Raccoupled adherens junction assembly. Both EDG-1-and EDG-3-regulated signaling pathways are required for endothelial cell morphogenesis into capillary-like networks. Indeed, SPP synergized with polypeptide angiogenic growth factors in the formation of mature neovessels in vivo. These data define SPP as a novel regulator of angiogenesis.

Phorbol 12-myristate 13-acetate activates rabbit neutrophils without an apparent rise in the level of intracellular free calcium

The addition of low concentrations of phorbol 12-myristate 13-acetate to rabbit neutrophils induces cell aggregation, degranulation, increased oxygen consumption and an increase in the amount of actin associated with the cytoskeleton without a rise in the level of intracellular free calcium as measured using the fluorescent probe quin-2. The ability of phorbol 12-myristate 13-acetate to initiate neutrophil responses similar to those produced by the chemotactic factor without causing a rise in the level of intracellular free calcium suggests two possibilities; that there is a second messenger in addition to calcium or that it activates the cells at a point distal to calcium mobilization. The possible role of diacylglycerol in neutrophil activation is discussed.

Direct demonstration of increased intracellular concentration of free calcium in rabbit and human neutrophils following stimulation by chemotactic factor

An increase in the level of intracellular free calcium concentration in rabbit and human neutrophils stimulated by chemotactic factors has been demonstrated directly using the calcium-sensitive fluorescent probe quin-2. Addition of f-Met-Leu-Phe (10(-9) M), C5a (3 x 10(-9) M) or leukotriene B4 (6 x 10(-8) M) to the neutrophils induces a rapid increase in the intracellular concentration of free calcium that reaches a maximum value 15 seconds following stimulation. At concentrations of f-Met-Leu-Phe less than 10(-8) M the enhancement is dose dependent with an ED50 of 8 x 10(-11) M and is significantly reduced in the presence of EGTA in the suspending medium.

Chemotactic factor causes rapid decreases in phosphatidylinositol,4,5-bisphosphate and phosphatidylinositol 4-monophosphate in rabbit neutrophils

Stimulation of rabbit neutrophils prelabeled with 32P by the synthetic chemotactic peptide f-Met-Leu-Phe induces a rapid decrease in the radioactivity in both phosphatidylinositol, 4,5 bis phosphate and phosphatidylinositol 4-monophosphate. The mean +/- standard error of the mean values of the maximum decrease in phosphatidylinositol, 4,5 bis phosphate occurred at 10 seconds following stimulation and is equal to 19 +/- 3% of the control value. The corresponding value for phosphatidylinositol 4-monophosphate occurred at 60 seconds following stimulation and is equal to 37 +/- 7% of the control value. On the other hand, the radioactivity in phosphatidic acid and lysophospholipids increased continuously with time following stimulation. The relationship of these changes to calcium release and neutrophil activation is discussed.

Membrane proteins related to water transport in human erythrocytes

CLASSICAL interpretations of the mechanism of water transport across mammalian red cell membranes assume the existence of aqueous membrane pores1–3. As the permeability coefficient to water measured under an osmotic pressure gradient is usually significantly higher than the corresponding value measured under diffusional flow, the human red cell membrane is thought to act both as a selective solvent and a molecular sieve. Its ability to function as a molecular sieve depends on the existence of the pores, which could be assembled from aggregates of integral membrane proteins which span the membrane4. It is generally agreed that at least two proteins span the human red cell membrane5–7. Using polyacrylamide gel electrophoresis we have found that a band which contains one of these proteins is selectively labelled by a water-transport inhibitor.

Tyrosine phosphorylation in human neutrophil

Protein tyrosine phosphorylation in human neutrophils was examined by immunoblotting with antibodies specific for phosphotyrosine. The addition of the human hormone granulocyte-macrophage colony stimulating factor to human neutrophils caused an increase in the tyrosine phosphorylation levels of several proteins. The increases in at least two of these proteins having molecular masses of 40 kDa (p40) and 54 kDa (p54) were rapid and were inhibited in pertussis toxin treated cells. The newly synthesized tyrosine kinase inhibitor ST 638 inhibited the increases in the levels of the tyrosine phosphorylation in p92, p78, p54 and p40 proteins. The epidermal growth factor receptor tyrosine kinase inhibitors were less effective. The addition of the chemotactic factor fMet-Leu-Phe to human neutrophils also caused an increase in tyrosine phosphorylation in some of these proteins. The pattern of the fMet-Leu-Phe-induced tyrosine phosphorylation was different from that produced by GM-CSF. The increases were also inhibited by ST 638. In addition, ST 638 inhibited superoxide production but not actin polymerization in control and GM-CSF-treated cells stimulated with fMet-Leu-Phe. Moreover, the active but not inactive phorbol esters increase the tyrosine phosphorylation only in the 40 kDa protein. These results suggest several points: (a) some of the responses produced by GM-CSF and fMet-Leu-Phe are mediated through tyrosine phosphorylation, (b) the GM-CSF receptor is coupled to a pertussis toxin sensitive G-protein, (c) the 40 kDa protein is probably the Gi alpha 2, and (d) the 78 or the 92 kDa protein is most likely the receptor for GM-CSF, which indicates that the receptor may have a tyrosine kinase domain.

Abnormalities in membrane micro viscosity and ion transport in genetic muscular dystrophy

WE have demonstrated recently several functional differences between membrane-bound enzymes from tissues of muscular dystrophic chicks and of controls1. Several other membrane anomalies have been associated with this disease2–7, but so far the only known biochemical anomaly in the affected membranes is a difference in lipid composition8,9. This could cause all the observed functional defects, including those in enzyme activities and permeability, through an alteration in the membrane microenvironment10. Certain physical properties of the microenvironment can be studied by fluorescent polarisation techniques that measure the mobility of a lipid-soluble fluorescent dye11,12. The basis for this approach is the fact that membrane cholesterol, which strongly affects the microviscosity of liposomes10, is present in significantly greater than normal amounts in genetic muscular dystrophy. Using this approach, we found (Table 1) that the membranes of muscle, liver and erythrocytes of dystrophic chicks have a significantly higher microviscosity than normal controls. The greatest difference was in muscle, the site of the clinical symptoms of this genetic disease. The higher cholesterol :phospholipid ratio characteristic of the diseased membrane is sufficient to account for the observed increase in microviscosity11.

Specific modulation of the intracellular pH of rabbit neutrophils by chemotactic factors

Abstract We have monitored the intracellular pH of rabbit neutrophils by following the distribution of the weak acid, 5,5-Dimethyloxazolidine-2, 4-dione. The synthetic formyl-methionyl chemotactic factors were found to induce complex and specific changes in the intracellular pH of the neutrophils, a rapid drop followed by a slower and more sustained rise. These effects are receptor mediated. The relationship of these events to the physiology of the neutrophils is discussed.

Arachidonic acid induced degranulation of rabbit peritoneal neutrophils.

Abstract We have found that arachidonic acid rapidly and selectively induces the release of lysosomal enzymes from cytochalasin B treated rabbit peritoneal neutrophils. 5, 8, 11, 14-eicosatetraynoic acid inhibits the arachidonate induced release with an apparent K D of 1.5 × 10 −6 M. 5,8,11,14-eicosatetraynoic acid (2.5 × 10 −5 M also inhibits the chemotactic factors and the A23187 induced release in the presence of cytochalasin B but does not affect the degranulation induced by A23187 alone. These observations strongly suggest a role for arachidonate metabolites in rabbit neutrophil physiology.

Calmodulin inhibitors block neutrophil degranulation at a step distal from the mobilization of calcium

Summary Two calmodulin inhibitors, trifluoroperazine and N-(6-aminohexyl-5-chloro-1-naphtalene sulfonamide, were found to be potent inhibitors of the chemotactic factor plus cytochalasin B induced lysosomal enzyme release from rabbit peritoneal neutrophils. On the other hand, these inhibitors did not affect the chemotactic factor induced intracellular calcium redistribution and membrane permeability increase. These results strongly suggest that calmodulin is involved in the expression of the various neutrophil functions. In addition, these compounds provide a unique experimental tool for the dissection of the excitation-secretion coupling sequence in neutrophils and other secretory cells.