Heterogeneity of mitochondrial matrix free Ca2+: resolution of Ca2+ dynamics in individual mitochondria in situ

The role of mitochondria in Ca2+ homeostasis is controversial. We employed the Ca2+-sensitive dye rhod 2 with novel, high temporal and spatial resolution imaging to evaluate changes in the matrix free Ca2+ concentration of individual mitochondria ([Ca2+](m)) in agonist-stimulated, primary cultured aortic myocytes. Stimulation with 10 mu M serotonin (5-HT) evoked modest cytosolic Ca2+ transients [cytosolic free Ca2+ concentration ([Ca2+](cyt)) 1,000 nM) and induced pronounced, reversible elevation of [Ca2+](m) (measured as rhod 2 fluorescence) in 60% of cells. This mitochondrial Ca2+ uptake usually lagged behind the cytosolic Ca2+ transient peak by 3-5 s, and [Ca2+], declined more slowly than did bulk [Ca2+](cyt). The uptake delay may prevent mitochondria from interfering with rapid signaling events while enhancing the mitochondrial response to large, long-duration elevations of [Ca2+](cyt). The responses of arterial myocytes to modest physiological stimulation do not, however, depend on such marked changes in [Ca2+](m)

Plasma membrane calcium pump-mediated calcium efflux and bulk cytosolic free calcium in cultured aortic smooth muscle cells from spontaneously hypertensive and Wistar-Kyoto normotensives rats

Objective To compare the efficacy of the calcium pump-mediated calcium efflux pathway in spontaneously hypertensive rats (SHR) with that in Wistar-Kyoto normotensive rats (WKY), at rest and after angiotensin II stimulation

Abstract P2-05-08: an assessment of the potential role of intracellular Ca2+store regulators in breast cancer cells

Background: Calcium (Ca2+) regulates many crucial cellular processes including cell survival, proliferation and death. Endoplasmic reticulum (ER) Ca2+ store levels are critical in the death-inducing effects of some anti-cancer agents. Modulators of ER Ca2+ signalling, such as neuronal calcium sensor-1 (NCS-1) may therefore represent new therapeutic opportunities to enhance the effect of some anti-cancer agents. NCS-1 is associated with poorer survival in breast cancer patients. However, the expression of NCS-1 in specific breast cancer molecular subtypes and its potential role in intracellular Ca2+ signalling in breast cancer cells has not been fully explored. Aim: To assess expression of NCS-1 in breast cancer molecular subtypes and assess the effect of silencing NCS-1 on intracellular Ca2+ homeostasis and on sensitivity to doxorubicin treatment in MDA-MB-231 breast cancer cells. Methods: NCS-1 levels were assessed in breast cancer molecular subtypes based on PAM50 subtyping using the TCGA public breast cancer database. Intracellular Ca2+ changes as a consequence of siRNA-mediated silencing of NCS-1 were evaluated using a Fluorescence Imaging Plate Reader (FLIPR) in MDA-MB-231 cells expressing the genetically-encoded Ca2+ indicator GCaMP6m. The effect of NCS-1 silencing on MDA-MB-231 cells treated with doxorubicin (0.03 and 1 μM, 24 h) was evaluated by cell nuclear enumeration (Hoechst 33342 staining) and the percentage of dead cells (propidium iodide staining). Images were acquired using an automated epifluorescence microscope (ImageXpress Micro). Results: Levels of NCS-1 were higher in the basal molecular subtype compared to other molecular subtypes. NCS-1 silencing promoted cell death induced by 1 μM doxorubicin. NCS-1 silencing had no major effect on cytosolic free Ca2+ levels as a result of either IP3-mediated Ca2+ store release with the purinergic receptor activator ATP or the protease activated receptor activator trypsin. However, NCS-1 silencing suppressed constitutive Ca2+ influx in MDA-MB-231 breast cancer cells. The expression of NCS-1 was positively correlated with the Ca2+ entry channel Orai1 in breast cancers on the TCGA database. Orai1 is associated with increased migration and invasiveness in some breast cancers

Elevated plasma membrane and sarcoplasmic reticulum Ca2+ pump mRNA levels in cultured aortic smooth muscle cells from spontaneously hypertensive rats

We have observed previously that Ca2+ pump-mediated Ca2+ efflux is elevated in cultured aortic smooth muscle cells from spontaneously hypertensive rats compared to those from Wistar-Kyoto rat controls. The objective of this work was to determine if these strains differ in mRNA levels for the PMCA1 isoform of the plasma membrane Ca2+-ATPase and the SERCA2 isoform of the sarcoplasmic reticulum Ca2+-ATPase. mRNA levels were compared in cultured aortic smooth muscle cells from 10-week-old male rats. PMCA1 and SERCA2 mRNA levels were elevated in SHR compared to WKY. Angiotensin II increased the level of PMCA1 and SERCA2 mRNA in both strains. These studies provide further evidence for alterered Ca2+ homeostasis in hypertension at the level of Ca2+ transporting ATPases in the spontaneously hypertensive rat model. These data are also consistent with the hypothesis that the expression of these two Ca2+ pumps may be linked. (C) 1997 Academic Pres

Abstract P1-19-05: establishment of a human neural progenitor cell microenvironment model to investigate signalling events in triple negative breast cancer brain metastases in a high-throughput setting

Background. Breast cancer brain metastases (BCBM) represents a terminal diagnosis accompanied by neurological decline and diminished quality of life. Average survival is less than a year following detection of neural lesions, due in part to an absence of targeted therapeutics. BCBM affects 12-17% of all breast cancer patients and over 25% of triple negative breast cancer (TNBC) patients. In the search for improved therapies, there is a focus increasingly on the role of the unique microenvironment of the brain in tumour progression. Few studies have investigated the establishment of an in vitro breast cancer brain microenvironment model that could enable the high-throughput identification and modulation of potential targets. In order to address this issue, a novel co-culture model was developed utilising the human neural progenitor cell (NPC) line ReNcell VM and MDA-MB-468 TNBC cells. Methods. ReNcell VM were differentiated as monolayers or neurospheres for four or ten days and stained for the glial marker glial fibrillary acidic protein (GFAP) and the neuronal marker β3-tubulin. Cultures were imaged via automated epi-fluorescence microscopy (ImageXpress Micro, Molecular Devices) and proportions of cell types were calculated with MetaXpress image analysis software. Differentiation characteristics of neurospheres and monolayers were compared. Functionality of neural cultures was determined using calcium assays with the muscarinic agonist carbachol as a validation tool. MDA-MB-468 cells loaded with a Cell Tracker Green dye were assessed for viability on the neural matrix via bright field and epi-fluorescence microscopy. Results. ReNcell VM cultured and differentiated for four days as neurospheres displayed a higher expression of β3-tubulin and GFAP than those cultured as monolayers (β3-tubulin 10.4% vs 7.7%, GFAP 53.6% vs 48.8%, respectively). Expression of β3 tubulin was higher after four days of differentiation than after ten days for neurosphere cultures (10.4% vs 4.4%, respectively). Carbachol elicited oscillatory calcium responses from ReNcell VM differentiated as neurospheres or monolayers, indicating functionality of both populations. MDA-MB-468 and ReNcell VM stained after four days of co-culture displayed successful integration of neoplastic cells on the neural matrix