Biomedical subjects

G J Bird

Publications and source records attributed to G J Bird.

4 recordsLinked to original sources

PubMed1998

Coherent backscatter enhances reflection confocal microscopy.

When thin optically transparent specimens are grown on reflective substrates, contrast in reflection confocal microscopy is markedly enhanced. This enhanced contrast allows for the visualization of the thin filopodia and organelles contained within the neuritic processes of PC12 cells in culture. The characteristics of this contrast enhancement suggest that it arises because of interference between light scattered from the specimen and coherently backscattered illumination reflected off the substrate. This technique provides a method for visualizing living cells or other similarly transparent objects on opaque substrates in a nondestructive manner.

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PubMed1995

The Ca(2+)-mobilizing actions of a Jurkat cell extract on mammalian cells and Xenopus laevis oocytes.

Randriamampita and Tsien (Randriamampita, C., and Tsien, R. Y. (1993) Nature 364, 809-814) suggested that an acid-extracted fraction from a Jurkat cell line contains a messenger responsible for the coupling of calcium entry to the depletion of intracellular stores, i.e. capacitative calcium entry. We found that the extract, prepared as described by Randriamampita and Tsien, caused Ca2+ entry in 1321N1 astrocytoma cells which was not blocked by the D-myo-1,4,5-trisphosphate-receptor antagonist, heparin. In contrast to astrocytoma cells, when applied to mouse lacrimal acinar cells and rat hepatocytes the Jurkat extract always caused the release of intracellular Ca2+, followed by Ca2+ entry across the plasma membrane. This activity of the extract on lacrimal cells was blocked by either intracellular injection of heparin or extracellular atropine. Similarly prepared lacrimal cell extracts gave Ca2+ responses when applied to astrocytoma cells or lacrimal cells which were similar to those for Jurkat-derived extract. However, extracts from hepatocytes had no effect. In most Xenopus oocytes, the Jurkat extract had no effect, while in a few oocytes, the extract gave a [Ca2+]i response similar to that seen in lacrimal cells, that is, release of Ca2+ followed by Ca2+ entry. We conclude that the actions of the Jurkat cell extract are not consistent with its containing the long sought messenger for capacitative calcium entry. It is likely that this fraction contains a number of factors that mediate Ca2+ response in different cell types, possibly through receptor-mediated mechanisms.

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PubMed1992

Functional homogeneity of the non-mitochondrial Ca2+ pool in intact mouse lacrimal acinar cells.

In the absence of extracellular Ca2+, treatment of mouse lacrimal acinar cells with maximal concentrations of methacholine released Ca2+ from intracellular stores. No additional Ca2+ was mobilized by subsequent application of the intracellular Ca(2+)-ATPase inhibitor, thapsigargin, the stable inositol 1,4,5-trisphosphate ((1,4,5)IP3) analog, inositol 2,4,5-trisphosphate ((2,4,5)IP3) (by microinjection), or the Ca2+ ionophore, ionomycin. However, following prolonged activation of cells by methacholine in the presence of extracellular Ca2+, Ca2+ accumulated into a pool which was released by ionomycin but not by thapsigargin. This latter accumulation was blocked by prior microinjection of ruthenium red, indicating that it represents mitochondrial uptake. In saponin-permeabilized lacrimal cells, two Ca(2+)-sequestering pools were detected: (i) a ruthenium red-sensitive, thapsigargin-insensitive pool, presumed to be the mitochondria; and (ii) a ruthenium red-insensitive, thapsigargin-sensitive pool. Only the thapsigargin-sensitive pool accumulated Ca2+ at concentrations similar to those in unstimulated cells. The thapsigargin-sensitive Ca2+ pool was sensitive to (1,4,5)IP3; however, in contrast to findings in intact cells, only 44% of this pool was releasable by (1,4,5)IP3 or (2,4,5)IP3. These data indicate that, in intact lacrimal acinar cells, all exchangeable (ionomycin-sensitive) Ca2+ residues in a pool which responds homogeneously to agonists, (1,4,5)IP3, and thapsigargin. Prolonged elevation of [Ca2+]i results in Ca2+ accumulation into a second, ruthenium red-sensitive pool, presumably mitochondria. Finally, permeabilization of the cells fragments the non-mitochondrial pool, resulting in two pools, one sensitive and one insensitive to (1,4,5)IP3.

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PubMed1991

Relationship between the calcium-mobilizing action of inositol 1,4,5-trisphosphate in permeable AR4-2J cells and the estimated levels of inositol 1,4,5-trisphosphate in intact AR4-2J cells.

Various experimental strategies were employed in an effort to explain the previously reported [Horstman, Takemura & Putney (1988) J. Biol. Chem. 263, 15297-15303] paradoxically high levels of inositol 1,4,5-trisphosphate [(1,4,5)IP3] in resting and substance-P-stimulated AR4-2J cells. The concentration-effect curves for substance-P-induced [3H](1,4,5)IP3 formation in [3H]inositol-labelled cells and substance-P-induced increase in intracellular [Ca2+] were essentially superimposable, suggesting that formation of (1,4,5)IP3 is limiting for cellular Ca2+ mobilization. In electrically permeabilized AR4-2J cells, (1,4,5)IP3 and other inositol polyphosphates stimulated Ca2+ release with potencies similar to those reported for other cell types, including the parent pancreatic acinar cell. Compartmentalization of basal (1,4,5)IP3 was suggested by the fact that this material was stable in the presence of antimycin A, although this toxin completely blocked agonist stimulation of phospholipase C. However, subcellular fractionation as well as permeabilization of the cells with Staphylococcus aureus alpha-toxin failed to provide evidence for binding or sequestration of [3H](1,4,5)IP3 in AR4-2J cells. The density of (1,4,5)IP3 receptors in AR4-2J cells was not sufficiently large to impose non-linearity in the relationship between (1,4,5)IP3 concentration and (1,4,5)IP3-induced Ca2+ release. Thus the apparent high concentrations of (1,4,5)IP3 in resting and stimulated AR4-2J cells are not indicative of atypically low sensitivity or high concentration of (1,4,5)IP3 receptors, nor is there evidence for compartmentalization of (1,4,5)IP3 outside of the cytoplasm in these cells. It is possible that soluble factors in the cytoplasm of AR4-2J cells regulate the free concentration of (1,4,5)IP3 or the sensitivity of receptors to (1,4,5)IP3.

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