Biomedical subjects

Tony J Collins

Publications and source records attributed to Tony J Collins.

6 recordsLinked to original sources

PubMed2004

Calmidazolium and arachidonate activate a calcium entry pathway that is distinct from store-operated calcium influx in HeLa cells.

Agonists that deplete intracellular Ca2+ stores also activate Ca2+ entry, although the mechanism by which store release and Ca2+ influx are linked is unclear. A potential mechanism involves 'store-operated channels' that respond to depletion of the intracellular Ca2+ pool. Although SOCE (store-operated Ca2+ entry) has been considered to be the principal route for Ca2+ entry during hormonal stimulation of non-electrically excitable cells, recent evidence has suggested that alternative pathways activated by metabolites such as arachidonic acid are responsible for physiological Ca2+ influx. It is not clear whether such messenger-activated pathways exist in all cells, whether they are truly distinct from SOCE and which metabolites are involved. In the present study, we demonstrate that HeLa cells express two pharmacologically and mechanistically distinct Ca2+ entry pathways. One is the ubiquitous SOCE route and the other is an arachidonate-sensitive non-SOCE. We show that both these Ca2+ entry pathways can provide long-lasting Ca2+ elevations, but that the channels are not the same, based on their differential sensitivity to 2-aminoethoxydiphenyl borate, LOE-908 [(R,S)-(3,4-dihydro-6,7-dimethoxy-isochinolin-1-yl)-2-phenyl-N,N-di[2-(2,3,4-trimethoxyphenyl)ethyl]acetamid mesylate] and gadolinium. In addition, non-SOCE and not SOCE was permeable to strontium. Furthermore, unlike SOCE, the non-SOCE pathway did not require store depletion and was not sensitive to displacement of the endoplasmic reticulum from the plasma membrane using jasplakinolide or ionomycin pretreatment. These pathways did not conduct Ca2+ simultaneously due to the dominant effect of arachidonate, which rapidly curtails SOCE and promotes Ca2+ influx via non-SOCE. Although non-SOCE could be activated by exogenous application of arachidonate, the most robust method for stimulation of this pathway was application of the widely used calmodulin antagonist calmidazolium, due to its ability to activate phospholipase A2.

Arachidonic Acid↗

PubMed2003

Local and global spontaneous calcium events regulate neurite outgrowth and onset of GABAergic phenotype during neural precursor differentiation.

Neural stem cells can generate in vitro progenitors of the three main cell lineages found in the CNS. The signaling pathways underlying the acquisition of differentiated phenotypes in these cells are poorly understood. Here we tested the hypothesis that Ca(2+) signaling controls differentiation of neural precursors. We found low-frequency global and local Ca(2+) transients occurring predominantly during early stages of differentiation. Spontaneous Ca(2+) signals in individual precursors were not synchronized with Ca(2+) transients in surrounding cells. Experimentally induced changes in the frequency of local Ca(2+) signals and global Ca(2+) rises correlated positively with neurite outgrowth and the onset of GABAergic neurotransmitter phenotype, respectively. NMDA receptor activity was critical for alterations in neuronal morphology but not for the timing of the acquisition of the neurotransmitter phenotype. Thus, spontaneous Ca(2+) signals are an intrinsic property of differentiating neurosphere-derived precursors. Their frequency may specify neuronal morphology and acquisition of neurotransmitter phenotype.

Animals↗

PubMed2003

2-Aminoethoxydiphenyl borate (2-APB) antagonises inositol 1,4,5-trisphosphate-induced calcium release, inhibits calcium pumps and has a use-dependent and slowly reversible action on store-operated calcium entry channels.

The action of 2-aminoethoxydiphenyl borate (2-APB) on Ca(2+) signalling in HeLa cells and cardiac myocytes was investigated. Consistent with other studies, we found that superfusion of cells with 2-APB rapidly inhibited inositol 1,4,5-trisphosphate (InsP(3))-mediated Ca(2+) release and store-operated Ca(2+) entry (SOC). In addition to abrogating hormone-evoked Ca(2+) responses, 2-APB could antagonise Ca(2+) signals evoked by a membrane permeant InsP(3) ester. 2-APB also slowed the recovery of intracellular Ca(2+) signals consistent with an effect on Ca(2+) ATPases. The inhibitory action of 2-APB on InsP(3) receptors (InsP(3)Rs), SOC channels and Ca(2+) pumps persisted for several minutes after washout of the compound. Application of 2-APB to unstimulated cells had no effect on subsequent Ca(2+) responses suggesting that it has a use-dependent action. Mitochondria in cells treated with 2-APB showed a rapid and slowly reversible swelling. 2-APB did not cause the mitochondria to depolarise, but it reduced the extent of mitochondrial calcium uptake. Although 2-APB has been demonstrated not to affect voltage-operated Ca(2+) channels or ryanodine receptors, we found that it gave a concentration-dependent long-lasting inhibition of Ca(2+) signalling in electrically-stimulated cardiac myocytes, where InsP(3)Rs and SOC channels do not play a significant role. Our data suggest that 2-APB has multiple cellular targets, a use-dependent action, is difficult to reverse and may affect Ca(2+) signalling in cell types where InsP(3) and SOC are not active.

Boron Compounds↗

PubMed2003

Mitochondria are morphologically heterogeneous within cells.

Mitochondria play key roles in the life and death of cells. We investigated whether mitochondria represent morphologically continuous entities within single intact cells. Physical continuity of mitochondria was determined by three-dimensional reconstruction of fluorescence from mitochondrially targeted DsRed1 or tetra-methyl rhodamine ethyl ester (TMRE). The mitochondria of pancreatic acinar, porcine aortic endothelial (PAE) cells, COS-7 cells and SH-SY5Y cells and neocortical astrocytes all displayed heterogeneous distributions and were of varying sizes. In general, there was a denser aggregation of mitochondria in perinuclear positions than in the cell periphery, where individual isolated mitochondria could clearly be seen. DsRed1 was found to be highly mobile within the matrix of individual mitochondria, with an estimated linear diffusion rate of 1 micro m s(-1). High-intensity irradiation of subcellular regions bleached the fluorescence of mitochondrially targeted DsRed1, but did not cause the mitochondria to depolarise or fragment. A lack of rapid fluorescence-recovery-after-photobleaching (FRAP) of DsRed1 indicated lumenal discontinuity between mitochondria. We observed a slow (half-time approx. 20 min) recovery of DsRed1 fluorescence within the irradiated area that was attributed to mitochondrial movement or fusion of unbleached and bleached organelles. Mitochondria were not electrically coupled, since typically only individual mitochondria were observed to depolarise following irradiation of TMRE-loaded cells. Our data indicate that the mitochondria within individual cells are morphologically heterogeneous and unconnected, thus allowing them to have distinct functional properties.

Animals↗

PubMed2002

Mitochondria are morphologically and functionally heterogeneous within cells.

We investigated whether mitochondria represent morphologically continuous and functionally homogenous entities within single intact cells. Physical continuity of mitochondria was determined by three-dimensional reconstruction of fluorescence from mitochondrially targeted DsRed1 or calcein. The mitochondria of HeLa, PAEC, COS-7, HUVEC, hepatocytes, cortical astrocytes and neuronal cells all displayed heterogeneous distributions and were of varying sizes. There was a denser aggregation of mitochondria in perinuclear positions than in the cell periphery, where individual isolated mitochondria could be seen clearly. Using fluorescence-recovery after photobleaching, we observed that DsRed1 and calcein were highly mobile within the matrix of individual mitochondria, and that mitochondria within a cell were not lumenally continuous. Mitochondria were not electrically coupled, since only individual mitochondria were observed to depolarize following irradiation of TMRE-loaded cells. Functional heterogeneity of mitochondria in single cells was observed with respect to membrane potential, sequestration of hormonally evoked cytosolic calcium signals and timing of permeability transition pore opening in response to tert-butyl hydroperoxide. Our data indicate that mitochondria within individual cells are morphologically heterogeneous and unconnected, allowing them to have distinct functional properties.

Animals↗

PubMed2002

2-aminoethoxydiphenyl borate (2-APB) is a reliable blocker of store-operated Ca2+ entry but an inconsistent inhibitor of InsP3-induced Ca2+ release.

Since its introduction to Ca2+ signaling in 1997, 2-aminoethoxydiphenyl borate (2-APB) has been used in many studies to probe for the involvement of inositol 1,4,5-trisphosphate receptors in the generation of Ca2+ signals. Due to reports of some nonspecific actions of 2-APB, and the fact that its principal antagonistic effect is on Ca2+ entry rather than Ca2+ release, this compound may not have the utility first suggested. However, 2-APB has thrown up some interesting results, particularly with respect to store-operated Ca2+ entry in nonexcitable cells. These data indicate that although it must be used with caution, 2-APB can be useful in probing certain aspects of Ca2+ signaling.

Animals↗