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A Rickard

Publications and source records attributed to A Rickard.

8 recordsLinked to original sources
PubMed2003

Measurement of the motility of endothelial cells in confluent monolayers.

OBJECTIVE: The reported experiments were designed to develop and test a method to measure the motility of endothelial cells in confluent cultures. METHODS: Endothelial cell cultures were recorded by time-lapse video. Frames from the recording were digitized, and cell locations were identified at 30-minute intervals using either the centroids or nuclei. The speed of the cells was determined for distances between their successive positions. Cells were stained immunocytochemically to determine the status of their adherens junctions. RESULTS: Prior to reaching confluence cells exhibited motility unhampered by collisions. After reaching confluence, cells continued to exhibit motility that decreased with time until the cells ceased effective movement. The motility in confluent monolayers persisted in the presence of complete adherens junctions. Motility could be restored in quiescent confluent cultures by the addition of fibroblast growth factor 2 (FGF-2). Generally, cells responding to FGF-2 moved without obvious loss of adherens junctions. CONCLUSION: These experiments provide evidence that (1) endothelial cells in culture are motile, (2) they do not form circumferential adherens junctions until the culture reaches confluency, (3) the establishment of adherens junctions itself does not interdict cell motion, and (4) the immotile state is reversible by FGF-2 in the presence of adherens junctions.

Adherens Junctions↗
PubMed2001

Stimulation of protease activated receptors on RT4 cells mediates arachidonic acid release via Ca2+ independent phospholipase A2.

PURPOSE: Protease activated receptors (PAR) represent a family of G protein coupled receptors with 7 membrane spanning domains that are activated by proteolysis of the N-terminus of the receptor by serine proteases. The presence of multiple PARs on the same cell is thought to extend the range of proteases a cell responds to rather than expand the range of intracellular responses. We investigated arachidonic acid and prostaglandin E2 release in the human urothelial carcinoma cell line RT4 in response to stimulation with thrombin, which activates PAR-1, and tryptase, which activates PAR-2. MATERIALS AND METHODS: RT4 cells were incubated with thrombin, tryptase or PAR agonist peptides and intracellular phospholipase A2 (PLA2) activity, arachidonic acid and prostaglandin E2 release were measured. Pretreatment with bromoenol lactone, a selective inhibitor for Ca2+ independent PLA2 (iPLA2), was also investigated. RESULTS: Thrombin and tryptase stimulation resulted in a 2 to 3-fold increase in membrane associated iPLA2 that was accompanied by comparative increases in arachidonic acid and prostaglandin E2 release. These responses were also observed when synthetic peptides representing the tethered ligand for each receptor were incubated with RT4 cells. Arachidonic acid and prostaglandin E2 release, and iPLA2 activation were completely inhibited by pretreatment with bromoenol lactone. CONCLUSIONS: Stimulating RT4 cells with PAR-1 or PAR-2 leads to the selective activation of iPLA2 as well as the release of arachidonic acid and prostaglandin E2, which may provide cytoprotection during an acute inflammatory reaction.

Arachidonic Acid↗
PubMed1999

Mast cell granule heparin proteoglycan induces lacunae in confluent endothelial cell monolayers.

The addition of rat mast cell granules to confluent bovine pulmonary artery endothelial cell monolayers resulted in the formation of numerous lacunae in the cultures. Several lines of evidence identified heparin proteoglycan as the component of the granule matrix responsible for the effect: presence of the activity in the proteoglycan fraction after chromatography of granule extracts, inhibition of granule activity by digestion with heparinase I, the failure of proteolysis of the proteoglycan fraction with proteinase K to significantly diminish its activity, and the failure of chymase and carboxypeptidase inhibitors to inhibit granule activity. The onset of hole formation was delayed for several hours after granule addition to the culture, and maximal hole formation occurred between 8 and 16 hours and was sustained as long as 24 hours. The lacunae formed by the separation of motile endothelial cells within the monolayer and was not attributable to cell contractile activity or cell loss. Time-lapse video recording showed that the holes were dynamic, individual holes expanding and regressing over a period of hours. Formation of lacunae occurred on gelatin and fibronectin surfaces alike. The presence of active chymase in the granules prevented the action of the proteoglycan. Heparin glycosaminoglycan as distinct from the proteoglycan did not similarly affect the endothelial monolayers but did block the action of granules added subsequently, indicating the likelihood of a heparin-reactive receptor or binding site.

Animals↗
PubMed1994

Eosinophil peroxidase accounts for most if not all of the peroxidase activity associated with isolated rat peritoneal mast cells.

Endogenous peroxidase has been reported in rat peritoneal mast cells and granules. Mast cell granules have also been shown to avidly bind exogenous eosinophil peroxidase. To examine the possibility that contaminating eosinophil peroxidase contributes to the reported rat mast cell peroxidase activity, mast cells were increasingly purified over sequential Percoll gradients. Such repeated centrifugations did not affect the histamine content of the cells or the secretory activity of cells, but the small increases in mast cell purity significantly reduced the specific activity of peroxidase; the remaining peroxidase activity of the mast cell fraction was in a range that could easily be accounted for by a small extent of contamination with eosinophils. An upper limit of 0.3 ng peroxidase/10(6) mast cells was determined from these measurements, ten times less than the values previously reported. When isolated mast cells were deliberately contaminated with soluble eosinophil peroxidase followed by granule isolation, the granules showed increased peroxidase activity, confirming the ability of mast cell granules to bind exogenous peroxidase.

Animals↗
PubMed1991

Rat mast cell tryptase.

Rat mast cell tryptase is located largely if not totally in the cell's secretory granules. When the active site reagent [3H]diisopropyl fluorophosphate was used to label tryptase and chymase simultaneously, the ratio of tryptase:chymase active sites was determined to be 0.05. In comparison to chymase and tryptase in other species and chymase in the rat, rat tryptase is poorly bound to the granule matrix as evidenced by (1) its release parallel to histamine on induction of secretion and (2) its appearance in the supernatant when isolated granules were stripped of their membranes with hypotonic medium. Tryptase on release from the granule is moderately stable at a pH of 5.0 but unstable at pH 7.5, the pH that the enzyme encounters on secretion from the cell. These several properties indicate that the role of rat mast cell tryptase extracellularly is likely to differ greatly from that of chymase.

Animals↗
PubMed1985

Effect on mast cell histamine of inhibiting histamine formation in vivo with alpha-fluoromethylhistidine.

An irreversible inhibitor of histidine decarboxylase, alpha-fluoromethylhistidine (FMH), was used to inhibit histamine formation by mast cells in vivo. Even at doses of FMH sufficient to reduce histamine formation more than 95%, the ability of mast cells to synthesize histamine recovered rapidly. It was possible, however, to sustain levels of histamine-forming activity below 10% of normal with continuous administration of FMH from subcutaneously implanted osmotic pumps. Administration of FMH under these conditions did not deplete significantly mast cell histamine but did prevent the increase in total mast cell histamine that occurs over 14 days and also prevented the reconstitution of mast cell histamine stores after depletion by treatment with polymyxin B.

Animals↗
PubMed1983

Evidence for control of mast cell granule protease in situ by low pH.

The second order rate constant, k2, for the inhibition of mast cell protease I by phenylmethanesulfonyl fluoride (PMSF) is lower for intact mast cells and isolated granules with intact membranes than for granules stripped of their membranes and suspended in medium at pH 7.1. In order to test the hypothesis that the decreased activity of the protease in intact granules is attributable to low pH, two agents capable of lowering pH in intracellular compartments similar to mast cell granules were tested. Ammonium chloride increased k2 of the protease in isolated granules with intact membranes and mast cells and wash out of the salt partially reversed this effect. Treatment of cells with nigericin also substantially increased the rate of protease inactivation by PMSF. These results are consistent with the proposal that the observed k2 is determined in whole or part by a low pH of the granule in situ or isolated with intact membranes. If the low k2 in situ is solely dependent on low pH, then the rate of protease inhibition can be utilized as an endogenous probe of granule pH. On this basis we have estimated the pH of the intracellular granule as 5.2 and that of the isolated granule with its membrane intact as 6.0. The value for the pH of granules in situ is lower than that previously estimated, and we have considered possible bases for this discrepancy.

Ammonium Chloride↗